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Cytoskeletal Intermediate Filaments

Part 1: Cytoskeletal Intermediate Filaments

00:00:07.14 Hello, my name is Bob Goldman
00:00:09.10 and I'd like to speak to you today about intermediate filaments.
00:00:14.16 Intermediate filaments are found both as complex networks
00:00:18.08 in the cytoplasm and in the nucleus of vertebrate cells.
00:00:24.03 The naming of intermediate filaments may be of interest
00:00:27.01 to some of you as the name
00:00:29.02 is derived from the fact that they are midway in size
00:00:34.04 between microfilaments and microtubules,
00:00:37.12 so they have a diameter of 10 nanometers (nm).
00:00:40.13 So they were originally called 10-nm filaments
00:00:42.25 but now they're called intermediate filaments.
00:00:49.02 The intermediate protein family is complex
00:00:52.08 and in fact is encoded by one of the largest families of genes
00:00:56.18 in the entire human genome.
00:00:59.01 Typically, intermediate filaments have a
00:01:05.10 highly, structurally conserved central rod domain, here seen mainly in red,
00:01:12.11 and there are five major types of intermediate filaments.
00:01:15.29 The first four, here,
00:01:18.07 types I - IV, are found in the cytoplasm,
00:01:20.18 so those are the cytoskeletal intermediate filaments.
00:01:24.00 The characterization of filaments, structurally, is
00:01:26.26 based on their highly,
00:01:29.08 as I mentioned highly conserved central rod domain,
00:01:32.07 which is separated by these linker groups
00:01:35.21 called L1, L12, and L2.
00:01:40.01 And these separate the central rod into segments 1A, 1B, 2A, and 2B,
00:01:45.29 which are all highly alpha helical in nature.
00:01:52.02 The keratins, which are the first two types,
00:01:55.12 both the acidic keratins and basic keratins,
00:01:58.16 are complex because they form heterodimers
00:02:01.09 as the basic building block.
00:02:03.10 And these heterodimers then interact to form, ultimately, 10-nm filaments.
00:02:08.20 They're presently known to be over 50 keratin genes
00:02:13.29 in the human genome.
00:02:16.19 In the case of Type III intermediate filaments,
00:02:19.08 such as vimentin, desmin, peripherin,
00:02:22.02 and glial fibrillary acidic protein,
00:02:24.18 these form homopolymers.
00:02:27.11 And in fact,
00:02:28.13 vimentin will be the major topic for today
00:02:31.01 because it's the simplest type of intermediate filament protein,
00:02:34.28 having only one protein chain
00:02:37.28 which self-assembles into 10-nm filaments.
00:02:42.26 And then the Type IV intermediate filaments,
00:02:45.08 the last type in the cytoplasm,
00:02:48.04 consist of neurofilaments,
00:02:50.20 which are actually three protein chains
00:02:52.24 forming a complex mainly found in the nervous system
00:02:56.15 -- nerve cells of the brain and the spinal cord
00:02:59.10 and the peripheral nervous system --
00:03:01.07 and a protein called nestin,
00:03:03.06 which is expressed mainly during early development,
00:03:07.18 synemin, and alpha-internexin,
00:03:09.14 which are other forms of intermediate filament proteins
00:03:13.15 which also form complex polymers.
00:03:16.19 And finally, there's the Type V intermediate filaments,
00:03:19.17 which are the nuclear lamins,
00:03:20.23 which we will discuss in the second part,
00:03:24.23 or in the second lecture.
00:03:30.07 Within cells,
00:03:32.17 there's a remarkable complexity of intermediate filament networks.
00:03:37.13 Almost every cell type that one looks at has a different,
00:03:40.05 not only intermediate filament composition,
00:03:42.14 but also intermediate filament network formation.
00:03:45.26 You can see on the left a vimentin filament network
00:03:50.13 in endothelial cells with a
00:03:53.01 ring of filaments around the nucleus.
00:03:55.17 And then on the right you can see keratin
00:03:58.05 in an epithelial cell and you can see this
00:04:01.03 remarkably complex network radiating
00:04:03.15 all the way from the center of the cell,
00:04:05.24 where it surrounds the nucleus as a cage,
00:04:08.11 all the way to the cell surface.
00:04:12.28 Now, the functions of cytoskeletal intermediate filaments
00:04:15.26 are numerous and we won't have time to discuss all of them today,
00:04:20.06 but we'll discuss a few of them.
00:04:21.27 But let me summarize by saying they're
00:04:23.10 very important in the mechanical integrity of cells,
00:04:26.11 they're very important in the shape of cells,
00:04:29.16 they're involved in both shape determination and maintenance,
00:04:34.12 they're very involved in cytoskeletal crosstalk
00:04:37.05 and stability,
00:04:38.15 that is they frequently bridge between microtubules
00:04:42.02 and actin filaments,
00:04:43.29 they're involved in signal transduction,
00:04:46.16 adhesion -- both cell-cell adhesion
00:04:49.19 and cell-extracellular matrix adhesion,
00:04:52.29 they're involved in cell motility,
00:04:54.16 and they're involved in the positioning
00:04:55.19 and tethering of organelles within the cytoplasm.
00:05:01.18 So let me just briefly go over the
00:05:04.13 structural features of intermediate filaments
00:05:06.18 and how they're built in the cell,
00:05:08.11 how they're assembled.
00:05:10.04 So the typical intermediate filament
00:05:12.29 is made of a dimer,
00:05:14.16 so two protein chains,
00:05:18.13 for instance in the case of vimentin,
00:05:20.11 since it's a homopolymer, it makes homodimers
00:05:23.22 and two dimers interact to form a
00:05:27.00 head-to-tail assemblies
00:05:29.23 which are in exact parallel and in register.
00:05:33.15 So the central rod domains actually line up
00:05:37.04 precisely so that they form a coiled-coil
00:05:41.23 and then we have the tail at one end
00:05:43.25 and the head domain, or the N-terminus and the C-terminus,
00:05:48.09 at the opposite ends of the dimer.
00:05:52.11 And then we can see,
00:05:54.19 this is just a blowup of this type of schematic,
00:05:58.13 and you can see that so-called Helix 1 and Helix 2
00:06:03.23 are separated by this region in the center
00:06:07.26 called the Linker 12 (L12) region.
00:06:10.17 And then we have the C-terminus,
00:06:12.13 which is relatively unstructured
00:06:14.29 and then the N-terminus at the other end
00:06:17.26 which is also relatively unstructured
00:06:20.07 in the case of cytoskeletal intermediate filaments.
00:06:25.00 During polymerization of,
00:06:28.06 in this case, human vimentin,
00:06:30.12 or any form of vimentin
00:06:33.12 because it's a highly conserved protein,
00:06:35.21 we can see in the far left panel
00:06:40.14 the formation of dimers.
00:06:43.12 And the two globular regions at one of the dimer
00:06:46.09 actually are the C-terminal domains.
00:06:49.21 And if you then go the next step in assembly,
00:06:52.19 and this is all done in vitro using bacterially expressed protein,
00:06:57.00 you assemble what are called Unit Length Filaments (ULF).
00:07:00.24 These are actually dimers
00:07:04.05 which interact with each other
00:07:06.11 and there are 8 of them,
00:07:07.23 so there are 16 dimers which form a 32-protein chain
00:07:14.09 Unit Length Filament.
00:07:15.18 And these ULFs,
00:07:17.19 which are approximately 50 nm in length,
00:07:21.20 actually interact with each other in tandem
00:07:25.06 to make longer and longer filaments.
00:07:27.04 There's two interacting at the lower left side
00:07:30.06 of this slide during the assembly process.
00:07:33.15 And then finally they go,
00:07:34.24 once you get long linkages of ULF,
00:07:38.09 you then go through a radial compaction,
00:07:41.15 which you can see in the last panel here.
00:07:45.26 And then,
00:07:49.03 it can be seen even in a more schematic way
00:07:52.25 in this next slide,
00:07:53.29 where you have Unit Length Filaments
00:07:56.00 interacting with each other,
00:07:57.22 linking in tandem,
00:07:59.13 to form longer and longer
00:08:01.25 intermediate filaments,
00:08:03.09 which then go through a super compaction
00:08:05.15 to form mature intermediate filaments.
00:08:08.18 The other important point about filament structure
00:08:10.14 is that it's apolar because the dimers,
00:08:15.03 which are in parallel in a register
00:08:17.11 with respect to the two protein chains,
00:08:20.06 interact in a head-to-tail fashion
00:08:23.01 which is called longitudinal assembly of dimers,
00:08:26.02 and they also interact laterally
00:08:28.18 in an oppositely-oriented and staggered way
00:08:33.09 to that you wind up getting tetramers
00:08:35.17 with opposite polarity.
00:08:37.16 And building up this structure into a 10-nm filament
00:08:41.15 gives you what's called an apolar filament
00:08:43.22 because it doesn't have a plus end
00:08:45.26 and a minus end like microtubules do,
00:08:48.20 and like actin.
00:08:51.03 So, now I'd like to go on just to tell you
00:08:53.07 how we prepare intermediate filament proteins from cells.
00:08:58.24 And, essentially, if you look on the left panel
00:09:00.08 it just shows you a blowup of a region of a cell
00:09:03.09 which was fixed and stained with antibody.
00:09:06.07 It's a fibroblast
00:09:07.06 and you can see the complex vimentin network.
00:09:10.23 In the middle panel, this cell has been extracted
00:09:13.26 with a solution containing high salt,
00:09:18.21 detergent,
00:09:19.14 and DNase I.
00:09:21.22 The DNase I is used to degrade chromatin,
00:09:25.06 it also helps to remove actin
00:09:27.03 from the preparation,
00:09:28.25 and there are no microtubules in these preparations.
00:09:31.19 And if you look on the next panel, you'll see that,
00:09:35.12 at higher solution
00:09:36.24 -- higher magnification in the EM,
00:09:39.16 in the electron microscope --
00:09:42.09 that in fact, these are intermediate filaments
00:09:45.15 and there's virtually no other structure
00:09:47.13 in the former cytoplasm of these cells.
00:09:52.13 And on the right panel, you can see the major band,
00:09:55.07 which is vimentin, and just above that
00:09:57.08 there are the nuclear lamins, which we'll speak about later
00:10:01.18 or in the next talk.
00:10:03.28 And finally, at the top of the gel
00:10:05.03 there's several proteins which are associated
00:10:07.18 with intermediate filaments such as nestin,
00:10:10.00 in this case,
00:10:11.03 and also a protein called plectin.
00:10:14.18 Because intermediate filaments
00:10:17.12 are so easy to isolate,
00:10:19.25 and in fact because they are pelleted
00:10:23.08 at low speed and almost virtually all of the protein
00:10:27.02 (intermediate filament protein)
00:10:28.23 in the cells like fibroblasts can be pelleted
00:10:31.14 at low speed and virtually no
00:10:36.11 protein is detectable in the supernatant,
00:10:39.27 for many years intermediate filaments were thought
00:10:42.16 to be very stable polymers
00:10:45.08 inside of cells.
00:10:47.02 Another property that led to their,
00:10:50.08 or leads to the,
00:10:52.02 I think, misconception that they are very stable,
00:10:55.00 is the fact that in an old paper
00:10:59.10 by Paul Janmey many years ago,
00:11:02.12 he demonstrated that under
00:11:05.12 conditions of stress
00:11:08.08 and strain in vitro,
00:11:10.14 in fact intermediate filaments
00:11:12.19 remained undamaged as you can see in the panel
00:11:15.29 on the left,
00:11:17.07 whereas microtubules and actin filaments
00:11:20.06 ruptured at relatively low shear stress
00:11:23.05 in this preparation.
00:11:24.23 On the right side, you can see a drawing
00:11:27.26 from a recent article in the literature
00:11:31.09 which shows that intermediate filaments,
00:11:33.17 in addition to being
00:11:36.28 undamaged by high shear,
00:11:38.23 can actually be stretched
00:11:40.25 up to three-and-a-half times
00:11:42.21 their normal length.
00:11:43.21 And once they become stretched to this extent,
00:11:47.18 they become very difficult to break.
00:11:51.02 So in fact this is an atomic force microscope
00:11:56.24 depicting what happens to an intermediate filament
00:11:59.09 when you stretch it,
00:12:01.02 and basically this is called,
00:12:04.04 their properties are known as
00:12:06.02 strain hardening properties.
00:12:09.04 And, for these two reasons,
00:12:11.00 both the fact that they're very extensible,
00:12:13.23 they can become very rigid when stretched,
00:12:16.28 and that they seem to be very insoluble
00:12:19.03 in biochemical preparations,
00:12:21.00 they were thought for many years
00:12:24.01 to be less dynamic that their cytoskeletal counterparts.
00:12:29.02 So let me go on and tell you a little bit
00:12:30.07 about the dynamic properties of intermediate filaments.
00:12:33.20 So, when we turn to live cells,
00:12:38.13 we realize that intermediate filaments
00:12:42.01 are very dynamic in vivo.
00:12:44.06 And in fact if you do a photobleaching experiment
00:12:47.26 where you look at recovery
00:12:49.20 of photobleaching with FRAP analysis,
00:12:52.11 you can see on the left side
00:12:54.17 a cell that was microinjected with
00:12:56.03 rhodamine-conjugated vimentin
00:12:58.18 and then in the next panel (B),
00:13:01.11 immediately afterwards that fiber
00:13:04.29 was bleached, photobleached.
00:13:07.07 And then in C D and E
00:13:08.09 you can see the photobleach recovery coming back.
00:13:11.11 And if you analyze in the bottom panel,
00:13:13.29 the recovery of fluorescence,
00:13:16.22 you'll find that it's equal all along the length of the fiber
00:13:20.23 as it recovers, which indicates that, in fact,
00:13:23.17 the filaments are not polar,
00:13:25.11 that they can actually exchange subunits
00:13:27.23 all along their length.
00:13:30.20 Another aspect of intermediate filament dynamics
00:13:34.12 can be seen very dramatically
00:13:36.19 when cells are attaining a shape in culture.
00:13:40.00 So, if you take trypsinized cells,
00:13:42.24 which are rounded,
00:13:44.05 placed in suspension,
00:13:45.20 and then place them down
00:13:47.04 onto a glass coverslip or slide,
00:13:50.19 and then watch them during the spreading process
00:13:53.11 by fixing and staining at different times
00:13:55.27 up to three or four hours,
00:13:57.20 you will in the top slide,
00:13:59.10 a lot of very small spots or particles,
00:14:03.16 and these are enriched in vimentin.
00:14:05.27 These cells were fixed and stained
00:14:08.05 with vimentin antibody for immunofluorescence.
00:14:10.28 In the middle slide you can see,
00:14:13.28 in fact, that as the cell spreads,
00:14:17.10 small filaments or short filaments,
00:14:19.01 which we called squiggles,
00:14:20.19 form and then these squiggles appear
00:14:22.22 to link end-to-end as we saw in vitro
00:14:26.01 to form longer and longer filaments
00:14:27.21 until the fully formed mature network
00:14:29.13 is formed in spread cells.
00:14:32.10 Movies of this reveal how dynamic this process in fact is.
00:14:36.27 This is a later stage of spreading,
00:14:39.03 but if you watch the particles in the movie
00:14:42.10 and the short filaments or squiggles,
00:14:44.08 you'll see that they're very dynamic:
00:14:46.18 they're moving bidirectionally
00:14:48.05 towards the edge of the cell.
00:14:50.24 And they can both move
00:14:53.01 towards the nucleus and away from the nucleus
00:14:57.01 as you can see.
00:14:58.19 Now, the types of movements
00:15:00.14 and the speeds can be very fast,
00:15:02.29 up to 1.5-2 micrometers per second (um/s).
00:15:10.28 This indicated,
00:15:11.27 of course that they were probably moving
00:15:14.00 along structures like microtubules.
00:15:16.10 And in fact, those vimentin particles,
00:15:20.02 especially moving at high rates of speed,
00:15:23.00 can be colocalized
00:15:26.09 when you fix and stain using double immunofluorescence.
00:15:30.03 And you see here microtubules
00:15:31.20 in the middle in red and
00:15:33.14 vimentin on the left in green,
00:15:37.07 during early spreading.
00:15:39.13 And then
00:15:42.12 in the overlay on the right, on the upper set of pictures,
00:15:46.09 you will see that there's
00:15:48.17 a very nice overlap between the
00:15:51.02 vimentin particles and microtubules.
00:15:53.07 And in fact, if you look on the bottom two panels,
00:15:55.28 you'll see that most of the particles
00:15:59.06 colocalized with the molecular motor kinesin,
00:16:04.25 which of course is a microtubule-based motor.
00:16:09.05 And without giving you all the data,
00:16:10.26 I just want to summarize by saying
00:16:13.25 intermediate filament particles move bidirectionally
00:16:16.24 because in fact they're associated
00:16:19.27 with both dynein and kinesin.
00:16:22.09 So they can move both
00:16:23.27 towards the plus end and the minus end of microtubules,
00:16:26.25 and they move at relatively high rates of speed.
00:16:30.07 They seem to slow down when they form filaments,
00:16:33.05 and they continue moving until filaments meet each other,
00:16:37.06 short filaments, meet each other,
00:16:40.01 and then become longer filaments.
00:16:43.09 So mature IFs seem to be made
00:16:46.14 from particles, squiggles,
00:16:48.22 linking up to form long IF,
00:16:50.29 and depending on microtubules-based motors,
00:16:53.17 and in fact, there's evidence for microfilament-based
00:16:56.10 (or actin-based) motors
00:16:58.13 participating in this process.
00:17:01.00 Now that I've demonstrated that intermediate filaments
00:17:03.26 are dynamic,
00:17:05.03 let me go on to say that intermediate filaments
00:17:08.18 are also very important in cell shape.
00:17:12.13 And this is no better depicted than in the
00:17:16.11 Epithelial to Mesenchymal Transitions (EMT)
00:17:19.22 that take place both in early development
00:17:22.07 and in the conversion
00:17:24.07 of tumor cells to metastatic tumor cells,
00:17:27.02 or benign tumor cells to metastatic tumor cells.
00:17:30.02 And these changes in cell shape
00:17:32.18 are always accompanied
00:17:33.26 by an upregulation in vimentin expression.
00:17:38.02 Typically, epithelial cells, which you see on the left,
00:17:40.27 express keratin,
00:17:42.10 and when they go through the epithelial-mesenchymal transition
00:17:46.26 they begin to express, and
00:17:49.11 eventually vimentin is expressed to very, very high levels.
00:17:54.11 So vimentin expression is a hallmark
00:17:56.28 of the cytoskeletal changes that take place during this EMT.
00:18:03.08 On the next slide, we can show that vimentin
00:18:05.00 actually can induce changes
00:18:07.03 in epithelial cell shapes very rapidly.
00:18:10.01 So if we look at the top panel,
00:18:13.02 we can see in MCF7 cells,
00:18:14.24 which is a human breast epithelial cell,
00:18:16.24 which is round, there's a single isolated cell,
00:18:20.13 and then in the middle panel it's a cell
00:18:22.26 just observed two hours after it was microinjected
00:18:26.15 with bacterially-expressed vimentin
00:18:29.07 and then fixed and stained.
00:18:31.13 So in a very short period of time,
00:18:33.20 this cell changes its shape,
00:18:35.26 just due to the expression,
00:18:37.26 in the form of protein injection
00:18:41.06 into the cytoplasm of an MCF7 cell.
00:18:45.05 Eventually that cell will recover its rounded shape,
00:18:49.15 because in fact vimentin expression
00:18:51.22 can't be sustained.
00:18:53.13 On the bottom panel,
00:18:55.11 we can see vimentin on the left
00:18:57.26 and the result of silencing vimentin expression
00:19:01.26 using shRNA.
00:19:05.06 And you can see the cells that are silenced
00:19:07.17 by the green color,
00:19:09.21 which is using GFP as a marker in this case.
00:19:13.15 And you can see the cells that are not transfected, in fact,
00:19:16.18 are normally shaped,
00:19:21.08 like a mesenchymal cell,
00:19:23.18 and the ones that are transfected and silenced,
00:19:26.18 you can see have changed their overall shape
00:19:29.11 to an epithelial cell.
00:19:31.03 So it looks like both the reverse of the EMT,
00:19:35.03 the mesenchymal to epithelial transition,
00:19:38.13 and the EMT itself,
00:19:41.12 can take place
00:19:43.26 just by controlling vimentin expression.
00:19:47.10 So cell shape seems to be very dependent
00:19:49.25 in this case on the intermediate filament composition of cells.
00:19:53.15 I should mention that microtubules and actin
00:19:55.07 are not affected in these experiments.
00:19:58.28 Now I just want to mention that
00:20:00.16 not only do you get shape changes in epithelial cells
00:20:03.08 when you express vimentin in them,
00:20:08.29 but vimentin also increases epithelial cell motility.
00:20:13.00 So here we see in the upper left panel
00:20:16.28 vimentin being expressed in an epithelial cell colony,
00:20:20.18 this is an MCF7 colony
00:20:23.18 which you can see in phase contrast below,
00:20:25.17 and this is just a few hours after transfection,
00:20:28.11 you begin to see expression of vimentin
00:20:31.00 as you can see in green.
00:20:32.16 And 24 hours later,
00:20:35.00 you can still vimentin networks in these epithelial cells,
00:20:39.21 but now all of the cells
00:20:41.04 in the lower right panel
00:20:42.19 have moved away from the colony's edge.
00:20:46.08 So this tells us that vimentin expression
00:20:49.23 will alter the cell-cell adhesive properties,
00:20:53.13 the cell-substrate adhesive properties,
00:20:55.17 change the shape,
00:20:56.28 and cause the cells to migrate
00:20:59.19 away from the colony,
00:21:00.27 again showing the importance of vimentin in motility.
00:21:06.20 And finally,
00:21:08.16 I just want to emphasize
00:21:11.03 the requirement for vimentin
00:21:14.01 in motility
00:21:15.19 by looking at,
00:21:17.14 on the left, are normal vimentin-containing fibroblasts.
00:21:22.27 In the middle,
00:21:24.08 these are fibroblasts
00:21:25.27 which have no vimentin,
00:21:27.24 in fact they don't express any intermediate filaments,
00:21:30.11 they're from the original vimentin knockout mouse.
00:21:34.26 And if you look, you can still see in those cells in the middle,
00:21:38.16 lots of activity around the surface,
00:21:40.06 these are surface ruffles
00:21:42.08 but no directional movement.
00:21:44.14 They can't move anywhere.
00:21:46.06 And you can see on the bottom
00:21:47.15 of each of these movies,
00:21:50.00 you can see the tracks made by the cells
00:21:52.02 over a 10-12 hour period.
00:21:54.28 In the right panel,
00:21:55.16 all of the cells are normal,
00:21:59.29 expect for this one at the tip of my finger,
00:22:02.19 which in fact is a cell
00:22:05.25 that is expressing a dominant-negative
00:22:07.23 mutant of vimentin
00:22:09.12 which you can see at the top.
00:22:11.29 It's basically a small piece of vimentin
00:22:13.06 which contains the initiation site
00:22:16.25 of the coiled domain plus the N-terminus,
00:22:20.08 and this acts to disassemble filaments in cells.
00:22:23.04 And you notice that one cell that's expressing
00:22:26.23 this mutant protein
00:22:28.08 is the only one in the field,
00:22:29.25 virtually, that doesn't move,
00:22:31.07 as you can see by the tracks below.
00:22:35.01 We then decided to look
00:22:37.02 at vimentin intermediate filaments in moving cells.
00:22:39.19 What do they look like?
00:22:41.21 So we just take moving fibroblasts,
00:22:44.26 in case it can be mouse fibroblasts
00:22:48.05 or human fibroblasts,
00:22:50.00 they look virtually indistinguishable,
00:22:52.12 and you can see in the top left the typical
00:22:55.08 fan-shaped moving fibroblast.
00:22:58.23 You can see, only now looking at vimentin,
00:23:01.28 you can see vimentin particles
00:23:05.13 are present in the lamellipod region
00:23:09.01 and at the leading edge the lamellipodium contains,
00:23:11.29 in the upper right panel,
00:23:13.14 you'll see lots of closely packed vimentin particles.
00:23:18.06 But filaments only seem to be forming,
00:23:20.15 or assembling,
00:23:21.24 in the rear region of the lamellipodium.
00:23:26.01 But the protein is certain there
00:23:27.21 in a disassembled state.
00:23:30.23 Below, in the top panel,
00:23:34.04 the top three images,
00:23:36.13 you can see a serum starved fibroblast
00:23:41.17 with vimentin filaments radiating all the way out to the edge.
00:23:45.26 And then it's very easy to induce ruffling in such cells,
00:23:50.27 as has been done for many years,
00:23:54.09 in many laboratories,
00:23:55.28 just by adding serum back.
00:23:57.25 So you take serum deprived cells
00:23:59.25 -- the cells were starved for 48-72 hours --
00:24:02.27 vimentin typically goes all the way to the surface,
00:24:05.27 the cells don't move, they're very quiet,
00:24:09.00 but as soon as you add,
00:24:10.07 actually within 10-15 minutes
00:24:11.26 after you add serum back,
00:24:14.13 on the bottom panel you can see the rapid formation
00:24:18.17 of lamellipodia and you can see rapidly,
00:24:21.00 that the same configuration that we see in normal moving cells,
00:24:25.21 we can now see in these
00:24:26.26 serum add-back experiments,
00:24:29.05 where you see vimentin particles
00:24:31.04 enriched in the leading edge
00:24:33.07 and then sort of a gradient of filament formation
00:24:36.14 back towards the nucleus.
00:24:41.05 Then we asked the question:
00:24:42.08 does the disassembly and retraction
00:24:43.28 of vimentin intermediate filaments
00:24:46.12 following serum addition involve phosphorylation?
00:24:49.26 And to do this, we used a phospho-specific antibody
00:24:53.05 which we received from the Inagaki lab in Japan.
00:24:58.00 And you can see in the top three images on the left,
00:25:02.09 vimentin on the left is
00:25:06.12 stained with vimentin antibody,
00:25:08.00 in the middle it's an antibody
00:25:11.13 against phospho-serine 38 (pSer-38),
00:25:13.18 which is in the N-terminal domain of vimentin.
00:25:17.13 And on the right is an overlay,
00:25:19.16 which of course just gives you the same pattern.
00:25:22.03 On the bottom, we can see a cell which was
00:25:27.25 fixed and stained
00:25:29.26 within a very short period of time,
00:25:31.19 within a couple of minutes after we added serum back.
00:25:35.24 And you can see now in the middle panel,
00:25:38.09 vimentin is on the left,
00:25:39.20 the middle panel shows what the Ser38 phospho-specific antibody looks like,
00:25:46.02 and you can see now
00:25:47.08 that the entire cell lights up,
00:25:49.26 is fluorescent (all these are indirect immunofluorescence images),
00:25:53.21 on the right is the overlay,
00:25:55.07 but a blowup of the region seen in the first panel,
00:25:59.13 panel D on the left,
00:26:01.17 is seen over on the right side here
00:26:05.07 where you can see that actually it looks like
00:26:06.15 the vimentin is retracted back from the surface
00:26:09.23 and that it's left behind particles and short filaments.
00:26:17.05 And here we can see,
00:26:18.15 in an SDS-acrylamide gel
00:26:23.21 and a western blot,
00:26:26.02 that in fact phosphorylation,
00:26:28.06 within one minute after adding serum,
00:26:33.04 goes up very significantly: many, many fold.
00:26:36.25 If you look at the zero time versus one minute,
00:26:40.00 you'll see the vimentin is rapidly phosphorylated;
00:26:43.02 in fact it only takes seconds
00:26:45.03 to become hyperphosphorylated under these conditions.
00:26:48.12 So this suggests that vimentin phosphorylation
00:26:51.00 is involved
00:26:52.18 in the local disassembly
00:26:56.01 and retraction of filaments
00:26:57.22 away from the cell surface in these regions.
00:27:00.29 And I should mention that these are the regions
00:27:03.10 which seem to initiate lamellipodia formation.
00:27:07.22 We also used another trick
00:27:09.23 to look at vimentin phosphorylation,
00:27:12.25 and that is using a form
00:27:16.08 of the small GTPase signaling factor called Rac1,
00:27:22.28 which is a member of the Rho family of GTPases.
00:27:27.10 It's very important,
00:27:29.05 we know from the actin literature,
00:27:31.21 that this particular signaling molecule
00:27:35.23 is very important in reorganizing actin
00:27:38.05 in the lamellipodial region.
00:27:40.16 And to do these experiments we used the photoactivatable form of Rac1,
00:27:43.29 provided to us by Klaus Hahn
00:27:46.18 from the University of North Carolina.
00:27:48.18 And this photoactivable form of Rac1
00:27:51.16 will become active
00:27:55.15 only when you shine 458 nm light on it,
00:28:00.05 and when we expose cells
00:28:02.05 which are expressing this
00:28:05.06 to global irradiation,
00:28:07.22 that is we just irradiate the whole cell
00:28:10.12 or a whole culture dish,
00:28:11.17 you can see that in fact
00:28:14.13 the intermediate filaments seem to retract
00:28:17.07 back away from the cell surface
00:28:19.08 under these conditions,
00:28:20.23 as in fact lamellipodia form.
00:28:23.05 And this is accompanied, again,
00:28:26.11 by a huge increase in the phosphorylation of Ser38.
00:28:34.11 So normally, you grow the cells in the dark
00:28:36.02 and when you shine light on them
00:28:37.09 they activate this Rac1.
00:28:39.19 And Rac1, it turns out, binds to PAK,
00:28:43.01 and PAK is a kinase
00:28:44.21 which is known to phosphorylation Ser38
00:28:47.16 in the vimentin head domain.
00:28:50.07 On the upper left,
00:28:51.10 we see the result of
00:28:54.00 a local irradiation experiment
00:28:56.17 with Klaus Hahn's PA-Rac1.
00:28:59.15 This cell, if you look beginning
00:29:04.13 now, was serum starved
00:29:06.05 and then we photoactivated
00:29:08.12 that small region
00:29:10.16 which you see in the movie.
00:29:12.03 But the important thing here
00:29:13.21 is to look at the edge of the cell,
00:29:18.03 and you'll see that when vimentin pulls back,
00:29:21.15 now,
00:29:23.06 the ruffle in fact forms.
00:29:26.01 So vimentin seems to disassemble
00:29:28.01 and retract from the cell surface.
00:29:30.09 And you can see this
00:29:32.25 even in regions which have very thick cables
00:29:36.03 of vimentin near the cell surface.
00:29:37.09 In the second upper panel,
00:29:39.28 you can see actually it looks like a steel cable
00:29:42.15 unwinding
00:29:45.26 after photoactivation of Rac1 in this small region.
00:29:49.18 And as this unwinds,
00:29:52.03 you can see the formation of short filament
00:29:54.20 and squiggles which seem to spin off
00:29:56.29 from this cable,
00:29:58.11 and it's only then that we really start to see
00:30:02.28 active lamellipodia forming.
00:30:05.15 So, in both cases,
00:30:07.12 it looks like vimentin actually
00:30:09.15 is stabilizing the cell surface,
00:30:13.27 and only when you disassemble and dismantle it,
00:30:17.10 presumably by phosphorylation,
00:30:21.08 and disassembly, and retraction away from the cell surface,
00:30:24.15 do you get lamellipods forming.
00:30:27.29 Now we did one other series of experiments
00:30:31.08 to show the importance of vimentin.
00:30:33.14 And that is using a peptide
00:30:35.19 called the 2B2 vimentin peptide.
00:30:38.24 2B2 is a mimetic peptide
00:30:41.01 which is derived
00:30:43.25 from the 2B region of the central rod domain.
00:30:48.02 And with our colleagues
00:30:50.17 Harald Herrmann and Ueli Aebi, we discovered that,
00:30:54.06 in fact, if you expose vimentin filaments,
00:30:58.04 on the left side panel A,
00:31:00.18 to this peptide
00:31:03.01 and leave it for a relatively short period of time
00:31:06.23 in vitro (this is an in vitro experiment),
00:31:09.07 you disassemble intermediate filaments
00:31:11.10 into these short pieces
00:31:13.19 which in fact are unit length filaments.
00:31:18.11 So using this 2B2 peptide,
00:31:22.19 we carried out some microinjection experiments.
00:31:26.27 In the top panels are just a repeat
00:31:29.21 of what the peptide does to intermediate filaments:
00:31:32.21 disassembles them
00:31:34.19 into small pieces
00:31:37.15 which in fact are unit length filaments.
00:31:42.05 And if you look at cells following injection of this peptide,
00:31:46.09 you see on the lower left figure D,
00:31:52.02 a cell which was injected
00:31:54.25 which had been grown in the absence of serum,
00:32:00.02 you can see that, in fact,
00:32:01.04 after injection the filament network
00:32:05.14 is retracted back towards the nucleus
00:32:07.23 and there are many particles
00:32:09.20 and short filaments in the region
00:32:11.26 between the nucleus and the cell surface.
00:32:14.20 And in fact, you also induce lamellipodia to form,
00:32:17.22 as seen here by one of the leading edge markers,
00:32:23.04 Arp2/3, which is an actin-associated protein.
00:32:28.04 And here we see a control cell
00:32:30.27 which was injected with scrambled peptide,
00:32:32.28 and you can see there's no retraction
00:32:34.24 back of the network, there's no disassembly.
00:32:38.25 On the next slide, I just want to show you a movie
00:32:41.22 of a cell which was injected with a
00:32:45.01 relatively high concentration, 2 micrograms/mL,
00:32:48.20 of the 2B2 vimentin mimetic peptide
00:32:53.18 and the filaments retracted.
00:32:56.25 This was a GFP-tagged vimentin-expressing cell.
00:33:03.20 And you can see that the filaments were retracted back towards the nucleus.
00:33:07.11 And then we,
00:33:08.22 in the absence of serum,
00:33:11.05 we get basically uncontrolled ruffling all over the surface.
00:33:18.17 And this cell cannot move anywhere, it just sits there.
00:33:21.05 So this fits nicely with our vimentin-null cells,
00:33:24.15 where we saw ruffling all over the surface.
00:33:26.21 In this case,
00:33:27.07 all we did was to inject the 2B2 peptide
00:33:31.17 in the absence of serum.
00:33:34.28 And then we did another experiment
00:33:36.17 using very low concentration,
00:33:38.13 0.5 micrograms/mL in the injection buffer,
00:33:41.22 and this cell was injected
00:33:43.14 just to the left side of the nucleus,
00:33:46.17 and this cell was in low serum
00:33:51.28 (1.5% serum)
00:33:55.12 and it was not moving at all
00:33:57.18 and there was no obvious ruffling.
00:33:59.22 And we injected just to the left of the nucleus.
00:34:02.27 And when you do this,
00:34:04.11 you see at the point of injection, very close to that region,
00:34:07.29 we induced a ruffle.
00:34:09.09 And if you look carefully,
00:34:10.10 you can see that this cell even begins to migrate
00:34:13.14 in the direction of the ruffle,
00:34:14.22 up towards the left of the screen.
00:34:19.16 So in summary, these observations tell us
00:34:23.04 that vimentin intermediate filament assembly
00:34:25.28 and disassembly are required
00:34:27.22 for the proper mesenchymal/fibroblast-type cell locomotion.
00:34:35.14 The regional and local disassembly of intermediate filaments
00:34:38.16 into their structural building blocks,
00:34:40.20 both the particle and squiggles,
00:34:42.28 modulates the formation of lamellipodia.
00:34:46.11 And conversely,
00:34:47.22 the regional and local assembly of intermediate filament networks
00:34:51.16 appears to be involved in inhibiting the formation of lamellipodia,
00:34:54.29 and what this means to us is that
00:34:56.29 it actually acts as a brake or a stabilizer
00:34:59.27 to the cell surface.
00:35:02.07 Intermediate filament disassembly and assembly appears,
00:35:06.28 in turn, to be regulated by kinases and phosphatases,
00:35:10.22 many of which are involved in signal transduction.
00:35:15.29 I'd like to acknowledge and thank
00:35:18.03 all of the members of my own laboratory
00:35:20.03 who have contributed in recent years to these studies,
00:35:24.27 and our collaborators,
00:35:26.15 both here and overseas,
00:35:28.28 and especially to our granting agencies,
00:35:31.27 the National Institute of General Medical Sciences,
00:35:35.05 the National Cancer Institute,
00:35:37.04 and Hannah's Hope Foundation,
00:35:38.22 for supporting our work.
00:35:40.19 Thank you.

Part 2: Nuclear Lamins

00:00:07.09 Hello I'm Bob Goldman
00:00:09.15 and in our previous talk
00:00:11.01 I discussed the
00:00:13.07 cytoskeletal intermediate filament networks of cells
00:00:16.29 and now I'd like to introduce you
00:00:18.24 to the nuclear lamins,
00:00:20.00 which are the Type V intermediate filament proteins,
00:00:22.27 which many consider to be the building blocks
00:00:25.12 of nuclear architecture.
00:00:27.21 And first, right on this introductory slide,
00:00:30.00 you can see the lamin network,
00:00:33.25 both forming an intense ring
00:00:37.16 around the edge of the nucleus and
00:00:40.07 in fact forming, in lesser concentrations,
00:00:46.07 a network throughout the nucleoplasm.
00:00:49.29 As I mentioned previously,
00:00:52.17 nuclear lamins are Type V intermediate filament proteins.
00:00:56.29 They are encoded by three genes:
00:00:59.04 the nuclear lamin A gene,
00:01:00.24 B1,
00:01:02.01 and B2.
00:01:04.16 And lamin C is an alternative splice product
00:01:07.27 of the lamin A gene,
00:01:10.00 so we have both lamin A protein, lamin C,
00:01:13.18 lamin B1 and B2,
00:01:16.02 in the nucleus of most vertebrate cells.
00:01:20.01 Certainly human cells.
00:01:22.14 Let me just go through the
00:01:24.09 complexity of the intermediate filament processing,
00:01:27.05 post-translation processing.
00:01:28.28 Before I do, I just want to mention that there are,
00:01:32.05 in addition to the central rod domain,
00:01:34.07 which is slightly longer than the rod domains
00:01:36.28 of proteins like the vimentin cytoskeletal protein,
00:01:41.29 there is a Ig-fold motif
00:01:46.03 and also a nuclear localization signal (NLS)
00:01:48.21 in the C-terminus of the protein,
00:01:52.02 in addition to a very C-terminal CAAX box.
00:01:59.04 And it is this CAAX box
00:02:00.22 that goes through numerous modifications
00:02:03.29 during processing
00:02:08.00 from pre-lamin A to mature lamin A.
00:02:11.26 In particular, what I tell you is going to be true of lamin A,
00:02:15.14 but not 100% true of lamin B1 and B2,
00:02:19.04 and I'll tell you the difference in a minute.
00:02:21.05 And you'll see why, later in the lecture,
00:02:22.26 I'm going through this complex series
00:02:26.07 of changes that take place.
00:02:29.23 So the CAAX box is a signaling for an enzyme,
00:02:33.01 farnesyltransferase,
00:02:34.29 to transfer a farnesyl group lipid moiety
00:02:38.13 to the cysteine residue
00:02:40.22 in the C-terminus.
00:02:43.04 And this is a signal
00:02:45.02 for an endo-peptidase called the AAX endopeptidase,
00:02:49.03 which removes the three amino acids,
00:02:53.04 the AAX at the C-terminus.
00:02:56.13 And this is a signaling for the carboxymethylase
00:02:59.10 to then place a methyl group
00:03:03.07 on the cysteine residue.
00:03:04.24 So now you have a farnesylated,
00:03:06.13 methylated cysteine
00:03:09.10 at the C-terminus.
00:03:10.28 And then this is a signal for another enzyme,
00:03:14.23 another endopeptidase, Zmpste24,
00:03:19.05 which then cleaves 15 additional amino acids
00:03:23.02 upstream from the C-terminus.
00:03:26.27 So all of this processing goes on,
00:03:29.06 and then removes
00:03:32.09 the farnesylation site
00:03:34.03 at the C-terminus.
00:03:36.00 But in the case of lamin B1 and B2,
00:03:38.11 these are both permanently farnesylated,
00:03:40.24 and this is not in their mature form.
00:03:45.00 Now the lamins assemble
00:03:47.05 in the nucleus into a complex
00:03:49.25 series of structures,
00:03:50.24 but let me just tell you where
00:03:53.20 it resides in the nucleus,
00:03:55.21 as seen here in the electron microscope
00:03:58.15 just under the nuclear envelope.
00:04:01.29 And if we look in the next
00:04:03.05 image at high magnification,
00:04:05.24 the lamins you'll see are forming an interface
00:04:10.25 in a region called the lamina,
00:04:11.29 between peripheral heterochromatin
00:04:14.22 and the inner membrane of the nuclear envelope.
00:04:18.17 And here you can see it in this famous picture by Don Fawcett,
00:04:21.18 you can see the outer nuclear envelope membrane,
00:04:24.03 the inner membrane, the other membrane,
00:04:27.25 the inner membrane,
00:04:29.22 and this fuzzy, fibrous layer which is called
00:04:32.18 the nuclear lamina.
00:04:34.07 Here, beautifully seen.
00:04:35.26 Rarely seen in this thickened configuration.
00:04:39.08 But then you see, just inside
00:04:42.15 this lamina region,
00:04:46.07 the peripheral elements of chromatin.
00:04:51.04 So the nuclear lamina forms a molecular interface
00:04:54.26 between the inner nuclear envelope membrane
00:04:57.17 and peripheral chromatin,
00:05:00.07 peripheral elements of chromatin.
00:05:02.12 The lamins, it turns out,
00:05:03.23 are the major proteins of this lamina region.
00:05:09.17 Lamins, as is the case with vimentin,
00:05:13.12 form dimers
00:05:15.16 and these dimers
00:05:17.12 interact in a way that's quite
00:05:20.00 different from vimentin.
00:05:21.18 That is, they interact by what's called
00:05:24.07 head-to-tail assembly of dimers.
00:05:27.12 So, dimer-dimer interactions take place,
00:05:31.05 rather than the kind of lateral association
00:05:34.01 we see,
00:05:35.13 and longitudinal associations we see,
00:05:39.07 with vimentin.
00:05:40.08 Here, we form dimer-dimer interactions
00:05:42.21 into long chains called protofilaments,
00:05:45.07 which then aggregate laterally
00:05:47.14 to form higher-order structures,
00:05:49.14 ultimately resulting in the formation
00:05:52.14 of these beautiful structures
00:05:54.28 originally described in Xenopus oocyte nuclei
00:05:58.07 by Ueli Aebi,
00:05:59.17 where you see almost a lattice-work,
00:06:03.28 almost a square lattice-work,
00:06:06.22 of lamin filaments.
00:06:13.15 The nuclear lamins have
00:06:15.13 structural properties and functions
00:06:17.23 which are not dissimilar from the intermediate filaments
00:06:20.10 in the cytoplasm.
00:06:22.01 But, now we know
00:06:23.26 many more specific possible functions
00:06:26.19 for these proteins in the nucleus
00:06:29.04 and they certainly are major structural proteins of the lamina,
00:06:33.24 which is surrounding the edge of the nucleus.
00:06:36.27 They're also located throughout the nucleoplasm.
00:06:40.08 And we know that
00:06:41.27 they're determinants of nuclear size and shape.
00:06:45.01 We know that nuclear assembly
00:06:47.00 and disassembly is regulated,
00:06:48.26 to a great extent,
00:06:50.10 by the nuclear lamins.
00:06:51.15 This has been shown by numerous laboratories.
00:06:54.12 They are involved in
00:06:58.01 the formation of the mitotic spindle.
00:07:00.10 They're involved in DNA synthesis,
00:07:02.13 specifically at the chain elongation phase of synthesis.
00:07:05.25 They're involved in DNA damage repair.
00:07:08.24 They have a role in transcription,
00:07:10.28 especially RNA Pol II transcription.
00:07:13.14 They're important in cell proliferation
00:07:15.10 and senescence.
00:07:16.20 They provide structural support for the nuclear membranes
00:07:20.05 and also support and positioning
00:07:22.15 of nuclear pores.
00:07:24.12 And they provide chromatin anchorage and organization sites
00:07:29.07 in the interphase nucleus.
00:07:33.29 I want to tell you a few of these,
00:07:35.25 give you a few pieces of experimental evidence
00:07:38.20 that what I told you, at least some of the things I told you,
00:07:40.25 are correct.
00:07:42.07 And that is, if you design a
00:07:44.24 dominant-negative mutant of lamins,
00:07:47.19 as we did quite a few hears ago
00:07:49.15 by removing the head domain,
00:07:51.06 you can see in this second diagram here,
00:07:55.01 that we've removed the N-terminus
00:07:58.03 of lamin and then this interferes
00:08:01.06 with lamin assembly because if you take this protein,
00:08:03.19 express it in bacteria,
00:08:05.06 and this was human nuclear lamin A,
00:08:09.27 so we call this headless lamin A or deltaNLA,
00:08:13.21 and inject it into a cell which is expressing GFP,
00:08:18.06 so this is a live cell expressing
00:08:20.03 GFP lamin A,
00:08:21.24 on the far left you can see the cell before injection.
00:08:25.20 And the same cell 30 minutes later,
00:08:27.26 you can see that the lamins are beginning to form foci,
00:08:32.12 they actually disassemble at the nuclear surface
00:08:36.11 and form foci throughout the nucleoplasm
00:08:39.18 and become significantly misshapen
00:08:43.08 within one to one and a half hours
00:08:47.20 as you can see here on the right.
00:08:49.18 And we noted that under these conditions
00:08:52.11 where you get disruption of lamin organization,
00:08:54.29 you get not only lamin A disrupted in this case,
00:08:59.28 but also lamin B.
00:09:02.12 And lamin disruption also alters nuclear shape
00:09:07.02 and the mechanical integrity of the nucleus
00:09:09.17 and also stops DNA replication,
00:09:13.00 as I'll show you in a minute,
00:09:14.20 and RNA Pol II transcription.
00:09:17.28 In the case of DNA replication,
00:09:21.10 although we can show this in cultured cells
00:09:23.22 following microinjection of single cells,
00:09:26.12 it's much easier to use the Xenopus laevis
00:09:30.01 cell-free nuclear assembly extract
00:09:32.15 which you make from Xenopus eggs.
00:09:36.08 Basically what you do is to,
00:09:38.28 in order to do biochemical assays,
00:09:41.07 this allows you to make
00:09:42.19 hundreds of thousands of nuclei
00:09:44.08 very rapidly in the test tube.
00:09:46.13 And on the left side you can see sperm chromatin,
00:09:50.05 which was been denuded,
00:09:51.27 that is the sperm tail is removed
00:09:54.02 and the membrane and the head
00:09:56.09 of the sperm is removed
00:09:58.08 so that all you have is the condensed mass of chromatin,
00:10:00.14 and you put it into this interphase extract,
00:10:03.06 which is easily prepared by centrifuging Xenopus eggs,
00:10:08.01 and then you get nuclear envelope assembly
00:10:11.06 and decondensation of chromatin
00:10:13.08 around each of the sperm heads.
00:10:15.22 And this just shows nuclear lamin staining
00:10:17.29 around the edge.
00:10:18.25 In the case of Xenopus eggs,
00:10:22.17 this lamin is called lamin B3.
00:10:25.21 And using these nuclei,
00:10:27.11 as soon as they become enveloped by the lamins
00:10:31.18 and the lamina and the nuclear envelope,
00:10:33.20 they initiate DNA replication.
00:10:36.05 And if we take these nuclei at that time
00:10:39.07 and just expose them to the deltaNLA,
00:10:45.07 or deltaNLD, or deltaNLD3
00:10:48.22 from Xenopus, any of these will work,
00:10:50.19 it will disrupt nuclear lamin organization
00:10:53.19 in these in vitro assembled nuclei
00:10:56.15 seen over here on the left in the middle panel.
00:10:59.27 You can see that nuclei become misshapen
00:11:02.06 and the lamins tend to form aggregates,
00:11:05.07 as we showed in the microinjected cultured cells.
00:11:09.12 So, here we can make lots of nuclei
00:11:11.07 and you can see that the lamins
00:11:13.08 are disrupted into these aggregates
00:11:15.06 which appear throughout the nucleoplasm.
00:11:18.11 And at the same time, if you look at the incorporation of
00:11:21.16 P32-labeled ATP,
00:11:25.03 which is a precursor to looking
00:11:29.18 at DNA replication,
00:11:31.24 you can show that, in a dose-dependent way,
00:11:34.22 DNA replication is inhibited
00:11:38.21 and you can see that
00:11:41.13 throughout this series of agarose gels.
00:11:46.01 So DNA replication can be
00:11:47.10 inhibited in a dose-dependent way
00:11:49.16 by adding this dominant-negative mutant of lamin assembly,
00:11:53.00 and in fact you can
00:11:55.26 basically reduce the replication by as much as 95%
00:12:01.05 with this lamin mutant.
00:12:05.15 But you always have residual DNA replication
00:12:08.11 and it turns out that that's because
00:12:11.02 the initiation of replication can take place
00:12:13.13 but the chain elongation phase of DNA replication
00:12:17.17 is inhibited.
00:12:18.20 And this is totally recoverable,
00:12:20.04 because if you put these nuclei back into normal medium,
00:12:25.15 they'll begin to replicate their DNA.
00:12:27.26 And one of the features we look for
00:12:30.25 in these disrupted lamin nuclei
00:12:33.00 were changes in two replication factors:
00:12:35.23 one is replication factor C (RFC),
00:12:37.25 seen in this top series,
00:12:39.18 and the other is PCNA,
00:12:43.24 proliferating cell nuclear antigen,
00:12:46.05 which are both very important factors
00:12:49.08 in the chain elongation phase of replication,
00:12:52.15 and in fact the function of DNA polymerase delta.
00:12:56.12 And you can see here that
00:12:58.24 both of these factors seem to line up, or colocalize,
00:13:02.05 with the aggregates of lamins
00:13:06.00 that form upon disruption of the lamin network.
00:13:11.03 And this of course is
00:13:14.04 double-indirect immunofluorescence
00:13:16.08 as an assay.
00:13:17.16 On the top is the,
00:13:21.24 upper left and lower left
00:13:24.06 are the controls for these experiments.
00:13:28.15 If you then look at the regulation of transcription,
00:13:33.29 in this case RNA Pol II transcription in HeLa cells,
00:13:37.03 what we did was to microinject on the left here in panel E,
00:13:41.24 you can see what happens after injecting
00:13:45.02 this truncated form of lamin
00:13:47.19 in a nucleus that was fixed about a half hour after injection.
00:13:51.13 And this one on the right
00:13:54.06 was injected at a slightly earlier time.
00:13:57.19 And you can see in both cases,
00:13:58.23 there's disruption of the lamin organization,
00:14:01.19 but the one on the left is even more disrupted.
00:14:04.22 And then on the right
00:14:05.29 you can see the result of the same nucleus
00:14:08.10 which was injected and incorporated,
00:14:11.08 or exposed to BrUTP,
00:14:13.16 which is a probe for RNA synthesis.
00:14:17.00 So you can see that there's a graded response,
00:14:18.25 this is greatly reduced in BrUTP incorporation,
00:14:22.14 and this is less reduced in BrUTP incorporation.
00:14:26.20 And on the bottom you have third cell nucleus
00:14:30.24 which was not injected at all with this dominant-negative mutant.
00:14:34.24 So there is a great reduction
00:14:37.01 in RNA synthesis,
00:14:38.11 as indicated by BrU incorporation,
00:14:42.00 and on the bottom
00:14:43.22 it just shows that there's always residual BrU incorporation,
00:14:47.02 and it's always associated with nucleoli,
00:14:49.13 which you can see in the center panel in phase contrast,
00:14:52.20 which indicates that RNA,
00:14:56.12 actually the data show that RNA Pol I and Pol III
00:15:00.03 are both going on normally.
00:15:04.15 It's only Pol II that's inhibited.
00:15:07.20 And then we repeated this same experiment with transcriptionally active,
00:15:11.18 isolated nuclei from Xenopus embryos.
00:15:15.02 The Xenopus eggs were fertilized
00:15:17.23 and the embryos were allowed to develop
00:15:19.17 for 10 hours into the mid-blastula transition,
00:15:22.27 which is the time when RNA polymerase II
00:15:26.09 becomes most activated.
00:15:30.03 And you can see, in the panel C,
00:15:35.25 that the transcription of messenger RNA
00:15:38.19 is greatly reduced compared to D,
00:15:44.03 in the same gel.
00:15:45.16 These are agarose gels showing, again,
00:15:50.11 P32-UTP incorporation into RNA.
00:15:56.03 The band at the bottom of these gels
00:15:59.04 in fact is transfer RNA,
00:16:04.02 which is synthesized, of course,
00:16:05.26 throughout these early developmental stages.
00:16:09.20 And in the case of looking at nuclei,
00:16:12.26 the nuclei that were isolated,
00:16:14.18 you can again show that when deltaNLA or deltaNLB3
00:16:18.10 is incorporated into these nuclei in vitro,
00:16:23.04 you get disruptions of lamin and again,
00:16:26.07 the lower right shows that BrU
00:16:28.09 is not incorporated.
00:16:29.22 So it looks like transcriptional control
00:16:38.19 of RNA Pol II is,
00:16:40.15 in some way, regulated by a normal lamin network.
00:16:46.21 Now I'd like to turn our attention,
00:16:48.25 in the last part of this talk,
00:16:50.22 to what rare human diseases
00:16:55.03 have told us,
00:16:56.29 and in fact provided an enormous number
00:16:59.26 of unexpected insights
00:17:01.13 into nuclear lamin structure and function.
00:17:07.07 So it turns out that in the last decade,
00:17:09.05 or slightly over a decade,
00:17:11.25 a number of, in fact a rather enormous number
00:17:15.07 of diseases and mutations,
00:17:17.14 now somewhere between 300-400 mutations,
00:17:21.13 have been related to diseases
00:17:24.23 caused by changes in the nuclear lamin A gene.
00:17:29.22 So this remarkable number
00:17:32.08 began with the discovery of
00:17:35.14 Emery Dreifuss Muscular Dystrophy
00:17:38.01 about 12-13 years ago
00:17:40.29 by Gisele Bonne in France.
00:17:44.04 And it turned out that this was a point mutation
00:17:47.08 in the nuclear lamin A gene.
00:17:51.08 And this was followed by an enormous number of
00:17:54.21 discoveries of mutations in the nuclear lamin A gene,
00:17:58.02 probably making it the most mutated gene,
00:18:01.26 or one of the most mutated genes, in the entire human genome,
00:18:05.15 that causes many different diseases.
00:18:08.16 And included in these diseases,
00:18:10.10 as you can see on the left
00:18:12.18 is the human disease
00:18:15.25 Hutchinson Gilford Progeria Syndrome,
00:18:18.26 which is a premature aging disease of children.
00:18:22.06 There are also forms of diseases of
00:18:28.17 neuromuscular junctions called Charcolt-Marie-Tooth disease.
00:18:32.02 There are also various forms of muscular dystrophies and,
00:18:36.18 in fact, dilated cardiomyopathies,
00:18:39.24 lipodystrophies,
00:18:41.08 and numerous other types of diseases.
00:18:46.03 The mutations are located everywhere
00:18:49.15 along the protein chain
00:18:52.06 and a given disease can have numerous sites
00:18:55.20 in different regions.
00:18:58.18 And this is very hard to explain
00:19:02.28 in terms of the total picture,
00:19:04.20 but if we concentrate on one particular disease,
00:19:08.03 and I want to do that today,
00:19:10.20 and that is the disease
00:19:12.04 Hutchinson Gilford Progeria Syndrome,
00:19:15.03 the premature aging disease of children,
00:19:17.17 and the reason for concentrating on this
00:19:20.09 is it's certainly the most extensively studied of all of
00:19:23.20 the so-called nuclear laminopathies.
00:19:28.27 Hutchinson Gilford Progeria Syndrome
00:19:31.25 (or Progeria),
00:19:35.17 if one looks at the clinical phenotype,
00:19:38.03 you find that children are born normal looking
00:19:43.03 and typically begin to lose their hair
00:19:45.02 at 12-18 months.
00:19:47.00 This is the same young girl
00:19:49.28 progressing through the disease;
00:19:51.16 these three images are of the same patient with progeria.
00:19:56.14 And you can see the disease
00:19:59.03 usually begins with losing hair or becoming bald,
00:20:04.15 and then skin becomes mottles,
00:20:06.10 there's loss of subcutaneous fat,
00:20:08.17 the nose becomes pinched,
00:20:13.18 and there is abnormal tooth development
00:20:15.22 and premature loss of teeth.
00:20:18.03 Typically they're short in stature
00:20:19.26 and they rarely achieve a height of 100 cm.
00:20:24.22 They have terrible problems with
00:20:27.22 arteriosclerosis, osteoporosis, hip dysplasia.
00:20:32.09 Typically, they die at a very early age,
00:20:35.09 usually 13-14 years,
00:20:37.24 of heart attacks and strokes.
00:20:40.19 And on the lower right,
00:20:42.08 we see a typical occluded artery
00:20:47.24 from a progeria patient.
00:20:50.18 Now, most of the mutations,
00:20:54.13 the major cause of progeria
00:20:57.03 is a mutation which is a silent mutation, G608G,
00:21:02.12 which introduces a cryptic splice site,
00:21:06.18 or makes available a cryptic splice site.
00:21:11.11 And this results in the loss, in pre-lamin A,
00:21:14.25 of 50 amino acids
00:21:17.07 between the end of the C-terminus,
00:21:19.07 just before the CAAX box,
00:21:21.18 and 50 amino acids upstream.
00:21:25.18 And this produces a protein,
00:21:27.16 which is called progerin, and it's a heterozygous situation
00:21:30.29 so therefore there's one normal allele.
00:21:35.12 There's wild type lamin A as well as
00:21:37.24 the mutant lamin A expressed in these patients.
00:21:40.10 So because of this truncation,
00:21:43.14 the protein that's produced, progerin,
00:21:47.15 is in fact permanently farnesylated
00:21:50.03 because that first cleavage site
00:21:52.28 is retained normally,
00:21:55.11 but the second cleavage site,
00:22:00.07 which should remove the farnesylated, methylated C-terminus,
00:22:04.16 in fact is not available.
00:22:06.20 So the Zmpste24 can't work
00:22:08.20 and therefore you get a permanently farnesylated form
00:22:11.14 of mutant lamin A
00:22:13.26 working together with its
00:22:16.23 normal wild type allele.
00:22:18.05 That's the most prevalent form of progeria
00:22:20.24 and accounts for 70-80% of all the patients known.
00:22:24.13 There are other very rare mutations
00:22:27.06 in different regions of the protein,
00:22:29.09 but in fact this is the one that's most common.
00:22:32.09 And it turns out that,
00:22:34.00 years ago, we found that
00:22:36.25 if one looks at the nuclear lamins in these patients
00:22:42.23 in early passage skin fibroblasts,
00:22:47.29 here passage 6, 13, and 26
00:22:50.07 of the same patient cell,
00:22:52.09 and in fact, the nuclei
00:22:55.00 become progressively misshapen.
00:22:56.18 So, one of the hallmarks of progeria
00:22:58.24 that's been widely used
00:23:01.11 is the formation,
00:23:02.26 or the dysmorphology,
00:23:04.25 of the nucleus.
00:23:07.19 And these nuclei are called blebbed nuclei.
00:23:10.27 And you can see in the middle panel here,
00:23:13.16 panel F,
00:23:14.21 that in fact there's an increase
00:23:17.11 in the truncated form of protein.
00:23:19.03 This third band appears with a lamin A antibody
00:23:22.10 in a western blot
00:23:23.29 of a whole cell extract.
00:23:25.22 You can see normally,
00:23:29.22 at early passage 9 here,
00:23:31.12 that in fact you see lamin A and C,
00:23:34.14 with the same polyclonal antibody,
00:23:36.06 but as you pass the cells in culture
00:23:39.25 or they get older in culture,
00:23:41.07 you see the addition of this third band,
00:23:43.09 which is called progerin.
00:23:45.21 And one other point that I'd like to make is that
00:23:49.15 we noticed very early on in late passage cells
00:23:51.20 with highly blebbed nuclei,
00:23:56.10 that in fact there is a separation
00:23:58.05 between lamin A and B.
00:24:00.11 Here, lamin A is seen in red by immunofluorescence,
00:24:03.10 and the B type lamins,
00:24:06.01 or B1/B2, are seen in green.
00:24:10.14 And then if one looks in the electron microscope
00:24:12.27 at these blebbed nuclei,
00:24:15.21 on the far left you can see a typically misformed nucleus
00:24:21.08 of a progeria patient's
00:24:23.06 skin fibroblasts
00:24:24.24 and in the middle panel you can see that, in fact,
00:24:28.06 the lamina,
00:24:30.03 this very dense black line here,
00:24:33.20 is greatly thickened compared to a normal control,
00:24:38.05 here seen way over on the right.
00:24:41.28 And there's very little peripheral heterochromatin
00:24:44.18 in these nuclei,
00:24:45.24 in fact there seems to be a great reduction in heterochromatin
00:24:48.21 throughout the nucleus.
00:24:51.13 So we turned to female cells,
00:24:54.13 both normal female and progeria cells,
00:24:57.12 to show losses in heterochromatin
00:25:00.23 because of the presence of the inactive X chromosome
00:25:03.25 seen on the upper left panel
00:25:07.07 stained with a histone mark:
00:25:10.00 histone H3,
00:25:11.29 lysine 27,
00:25:13.15 trimethylated (H3K27m3).
00:25:15.17 And the same mark you can see disappearing
00:25:17.28 as a function of passage number
00:25:20.22 in progeria cells in the lower left two panels.
00:25:24.23 In the middle it shows XIST RNA,
00:25:26.18 another mark for the
00:25:30.08 human inactive X chromosome,
00:25:32.26 and you can see as you go down
00:25:35.08 through these panels that, in fact,
00:25:38.11 the XIST RNA seems to be retained,
00:25:40.29 but it sort of unravels.
00:25:42.21 It's not as tight a structure
00:25:45.12 and appears to move interior into the nucleus.
00:25:49.16 And over on the right it just shows you these at higher magnification.
00:25:53.23 On the lower right, you can see the result of
00:25:57.18 an RT-PCR analysis,
00:25:59.11 which shows that the enzyme EZH2,
00:26:01.21 which is responsible for the H3K27 methylation,
00:26:08.13 is greatly reduced in amount
00:26:10.21 as a function of passage number, again.
00:26:14.03 And then on the left,
00:26:15.24 in the lower left,
00:26:17.28 we see that EZH2 is also greatly reduced
00:26:21.26 in the progeria cells
00:26:24.24 at later passages.
00:26:28.06 And on the right you can just see
00:26:30.00 another histone mark: H3K9me3,
00:26:36.04 also greatly reduced by western blotting
00:26:38.02 in progeria nuclei
00:26:40.02 at later passages
00:26:41.10 and you can also see
00:26:43.05 in the nuclei of the patients
00:26:45.00 that there's very little peripheral heterochromatin
00:26:48.16 containing the H3K9me3 mark, compared to controls.
00:26:56.17 So there are profound changes in heterochromatin in progeria,
00:27:00.01 and in fact this,
00:27:02.17 taken together with the
00:27:04.16 finding that progerin
00:27:07.24 is permanently farnesylated
00:27:11.04 and in fact seems to be
00:27:13.10 a dominant-negative effects in these patients,
00:27:18.14 this led to a very rapid,
00:27:20.22 probably one of the fastest bench-to-bedside
00:27:23.24 drug trials in the history of medicine
00:27:26.23 because there were available
00:27:28.16 farnesyl transferase inhibitors.
00:27:33.04 And this attracted participants from all over the world;
00:27:36.17 parents brought their kids,
00:27:38.06 and are still bringing their children,
00:27:40.14 progeria patients,
00:27:43.06 to Boston Children's Hospital where these trials
00:27:46.07 are being conducted.
00:27:48.07 In turns out that when you
00:27:49.28 inhibit farnesylation of progerin
00:27:52.29 by adding FTIs in the nuclei of patient cells,
00:27:56.26 as was done originally by the Capell group in Francis Collins' lab,
00:28:03.01 you can see that there's a reversal,
00:28:04.21 on the lower left,
00:28:05.24 of the nuclear morphology,
00:28:08.26 that is the nuclei look more normal
00:28:11.13 after farnesyl transferase inhibitor treatment.
00:28:18.03 And the nuclei in progeria cells
00:28:19.22 also showed a rather remarkable separation
00:28:24.13 of the A and B type lamins.
00:28:26.06 I showed this picture earlier, but here it is at higher magnification,
00:28:30.02 and you can see in red lamin A
00:28:32.16 and you can see the B type lamins
00:28:35.02 are excluded from these large blebs
00:28:38.06 but are present in the other non-blebbed region
00:28:41.17 of the nuclear surface.
00:28:46.11 So we wanted to know how
00:28:48.17 this change
00:28:50.29 in nuclear lamin structure
00:28:53.13 might be achieved.
00:28:54.17 And to do this, we just carried out some
00:28:56.23 typical cell biological experiments,
00:28:59.13 which involved silencing each of the lamins.
00:29:02.04 So we designed silencing vectors,
00:29:05.19 shRNAs,
00:29:07.14 for lamin A/C,
00:29:08.27 for lamin B1,
00:29:10.08 and lamin B2,
00:29:12.24 and you can see in the top panel here, using western blots,
00:29:16.15 that these worked quite efficiently,
00:29:19.12 and the controls are on the right in each case
00:29:22.11 and the silenced cells are on the left.
00:29:27.16 And in all cases in these experiments,
00:29:29.18 we used HeLa cells.
00:29:32.07 And on the left you can see
00:29:35.03 lamin A/C staining in a lamin B1-silenced nucleus,
00:29:39.25 and you'll notice that there are blebs
00:29:41.25 in the silenced nuclei.
00:29:43.20 The red nuclei in the middle are unsilenced cells,
00:29:47.10 this was done by transient transfection
00:29:49.18 so we always had some unsilenced cells,
00:29:52.07 and when you have these you see normal lamin B1.
00:29:55.28 But in the case of the silenced nuclei,
00:29:59.23 invariably we saw blebbing at the surface.
00:30:03.05 And this was not true when we
00:30:08.29 silenced A or lamin B2.
00:30:11.14 Only lamin B1.
00:30:14.29 The blebs were induced
00:30:18.13 and we found that they were mainly lamin A enriched.
00:30:21.16 That is, they were devoid of the other lamin,
00:30:24.10 lamin B2,
00:30:25.18 because we knew LB1 was missing in these cells.
00:30:29.17 So LB1 silencing leads to the formation of
00:30:31.28 lamin A-rich blebs,
00:30:34.07 which looked very, very similar
00:30:39.08 to the progeria situation.
00:30:43.11 Then we did some experiments just to begin to see if we
00:30:47.19 could induce histone modifications.
00:30:49.19 And the answer is that in the blebs,
00:30:52.02 we saw a great reduction,
00:30:54.04 that is in these lamin-A rich blebs,
00:30:56.11 we saw a great reduction
00:30:57.29 in histone marks
00:31:00.15 that usually stain peripheral heterochromatin,
00:31:03.11 in this case H3K9, in the bottom panel, -me2,
00:31:09.26 was greatly reduced
00:31:11.13 and almost not detectable in the blebs
00:31:13.14 relative to the main nuclear body,
00:31:16.05 which you can see towards the bottom of these images.
00:31:18.23 Again, this is indirect immunofluorescence throughout.
00:31:24.00 And then we asked questions
00:31:26.15 about the blebs
00:31:29.11 which had to do with their function
00:31:31.19 or the chromatin related to these blebs,
00:31:35.09 what were their properties?
00:31:37.03 And if you look at the upper left panel,
00:31:41.06 where it says DNA in A,
00:31:43.12 you'll notice that the bleb that's present,
00:31:46.19 this is the same nucleus all across the top,
00:31:49.27 is greatly reduced with respect to DNA concentration.
00:31:54.22 This is a Hoechst stain,
00:31:57.07 and you can see a great reduction in chromatin,
00:32:00.27 indicating that there's euchromatin in the bleb
00:32:03.16 and not heterochromatin.
00:32:05.23 So then we looked at RNA polymerase II,
00:32:09.26 and if you look at the inactive form of Pol II,
00:32:12.29 you find it's distributed throughout the nucleus
00:32:15.14 and even in the bleb,
00:32:19.18 but then when you look Pol II-0,
00:32:21.07 an active form of Pol II,
00:32:24.29 it's a specific antibody,
00:32:27.27 you find that it seems to be concentrated in the blebbed region.
00:32:32.15 And then we just asked the question,
00:32:34.16 well since Pol II-0 is concentrated in the bleb,
00:32:38.18 we said: maybe it's really transcriptionally active?
00:32:41.18 So, again we used the BrUTP incorporation
00:32:44.16 to look at RNA synthesis in blebs
00:32:48.11 and if you look at that third row of images,
00:32:52.22 you'll see that BrU is greatly reduced,
00:32:55.22 barely detectable in the bleb,
00:32:57.16 whereas it is detectable in the rest of the nucleus.
00:33:01.06 And then if we look,
00:33:02.07 and you can see LB1 is of course silenced in all of these,
00:33:06.20 occasionally a nucleolus
00:33:08.08 would get trapped in a bleb,
00:33:09.27 you can see in the bottom panel,
00:33:11.16 and there you can see that brighter object in the bleb,
00:33:14.28 in fact, is a nucleolus,
00:33:17.20 because the ribosomal RNA continues to work
00:33:22.17 in the presence of lamin A.
00:33:25.06 So there's no inhibition of ribosomal RNA synthesis.
00:33:28.29 And then we looked at a couple of other marks for transcription by Pol II,
00:33:33.20 and that's H3K4me3 and H3K36me3,
00:33:38.24 and we found that K4me3 is present in the bleb,
00:33:44.14 but K36me3 seems to be absent in the bleb.
00:33:48.10 And finally, we came to the conclusion,
00:33:53.23 a situation that we're still working it,
00:33:55.29 and that is, it looks like in the bleb,
00:33:58.17 RNA Pol II can be initiated,
00:34:01.13 polymerase action can be initiated,
00:34:04.08 but it can't be completed.
00:34:05.27 So it looks like a phenomenon called
00:34:07.28 RNA polymerase stalling,
00:34:10.11 or Pol II promoter proximal stalling.
00:34:13.18 So, maybe it looks like initiation of RNA synthesis can take place,
00:34:18.02 mRNA synthesis,
00:34:19.17 but the elongation phase can't take place in this case.
00:34:24.01 So because of the changes in,
00:34:28.09 and differences in silencing of lamin B1 and B2,
00:34:33.21 we started to take a more serious look at the different,
00:34:36.22 at the possibility
00:34:38.06 that A and B type lamins really have different functions.
00:34:42.11 And what we had noticed earlier,
00:34:45.02 in our progeria experiments,
00:34:47.09 and here on the left you can see western blots of
00:34:51.29 another mutation causing progeria,
00:34:55.11 which is E145K,
00:34:58.06 you can see that lamin B1 is greatly reduced
00:35:02.11 when the cells become prematurely senescent in culture.
00:35:05.24 And that is one of the hallmarks of progeria:
00:35:08.12 cells don't grow that well in culture.
00:35:10.14 Progeria patient cells will grow a
00:35:12.23 limited number of generations,
00:35:15.03 both with the more common G608G mutation
00:35:18.09 and this E145K mutation,
00:35:21.03 we saw premature senescence,
00:35:24.05 loss of growth,
00:35:25.16 and a reduction in LB1,
00:35:27.16 but if you look down on the left,
00:35:29.26 for both the progeria patient on the left, that panel,
00:35:37.24 you can see a great reduction in lamin B1.
00:35:41.09 Lamin B2 is reasonably retained,
00:35:43.28 and lamin A and C are also retained.
00:35:46.16 So we started asking questions
00:35:48.21 about lamin B1 during normal cell growth and senescence.
00:35:52.29 And here you can see the result of
00:35:56.28 just beginning to look at WI-38 cells,
00:35:59.26 which are a classical system,
00:36:01.15 cell system for looking
00:36:05.01 at the growth and senescence of normal human, diploid fibroblasts.
00:36:10.08 So, as I mentioned below,
00:36:12.09 to date we've focused most of our attention on lamin B1
00:36:15.12 because of our results from progeria.
00:36:18.14 And here you can see that the loss of lamin B1
00:36:20.07 is closely coupled to the appearance of senescent markers
00:36:24.18 in human diploid fibroblasts.
00:36:27.21 So here we have, beginning at passage 30,
00:36:30.27 and then we look at passage 39,
00:36:34.05 and then passage, doubling,
00:36:35.21 I should say, 41,
00:36:37.22 and you can see that the nuclei from progeria cells,
00:36:41.15 as we move across,
00:36:43.03 change in different ways.
00:36:46.09 For instance, at passage 30,
00:36:50.14 the lamins look quite normal.
00:36:52.16 Lamin B1, lamin B2, lamin A/C
00:36:55.18 have their normal distribution.
00:36:58.11 But by passage 39 and 41
00:37:01.01 we begin to see a loss in lamin B1.
00:37:03.23 And by 41,
00:37:05.22 you can see almost complete loss of lamin B1.
00:37:09.05 This is accompanied, in the lower panel toward the right,
00:37:14.12 you'll see a misshapen nucleus stained with LB2.
00:37:18.00 So, the nuclei get larger and somewhat misshapen.
00:37:21.06 And then as we move across,
00:37:23.13 these cells at passage doubling 41
00:37:25.28 are in fact senescent
00:37:27.20 and we see the appearance
00:37:29.26 of known senescence markers,
00:37:31.26 including SAHF, which is "SAHFs",
00:37:37.02 which are senescence-associated heterochromatic foci.
00:37:40.11 And we also see a loss of incorporation of BrdU,
00:37:44.16 which indicates that DNA synthesis
00:37:46.14 goes down to zero
00:37:48.24 in the senescent cells,
00:37:51.02 indicating no more growth.
00:37:53.00 And you can also see the increase,
00:37:55.25 up to 100% of the cells,
00:37:58.25 containing what's called senescence-associated beta-galactosidase.
00:38:04.03 All of these are markers for senescence,
00:38:08.22 and we'd like to add to that biomarker list lamin B1,
00:38:12.29 and this has been verified by several different laboratories now.
00:38:17.17 So, we went on to look at other,
00:38:20.18 first of all quantitative western blotting
00:38:22.22 of the loss of lamin B1,
00:38:25.24 which we can see in the upper panel here
00:38:29.20 as a function of time
00:38:31.05 as cells enter senescence, LB1 is lost.
00:38:33.28 But you can see,
00:38:35.19 on the right hand side here,
00:38:37.03 that LB1 is lost but LB2, LA, and LC
00:38:42.17 are in fact retained.
00:38:45.16 And the mRNA,
00:38:47.05 as measured by RT-PCR analysis for lamin B1 in red,
00:38:52.17 you can see in this histogram,
00:38:53.29 is greatly reduced even by passage doubling 39,
00:38:57.16 and by passage PD 41,
00:39:01.10 is almost impossible to detect.
00:39:03.29 So both the RNA and protein levels
00:39:06.25 of lamin B1
00:39:08.29 are greatly reduced in senescence.
00:39:11.28 LB1 silencing also slows
00:39:17.12 proliferation and induces premature senescence
00:39:20.23 in WI-38 cells, and we showed this
00:39:24.29 by taking relatively early passage cells,
00:39:27.25 let's say passage doubling 24 cells,
00:39:31.10 and if you look on the upper left
00:39:33.02 you'll see that LB1
00:39:35.16 is greatly reduced in the silenced cells,
00:39:40.11 and this is correlated with a loss of growth
00:39:44.12 as you can see here in this growth curve,
00:39:48.12 compared to control
00:39:51.25 which is scrambled sequence here
00:39:53.24 instead of the silencing vector.
00:39:55.21 And you can show also,
00:39:58.10 that in cells the nuclei get larger
00:40:02.24 and in fact look senescent
00:40:05.04 relative to scrambled controls,
00:40:06.27 but most importantly they show significant increase
00:40:10.01 in senescence-associated heterochromatic foci,
00:40:14.22 a significant loss of BrdU incorporation,
00:40:18.11 indicating that the cells are rapidly
00:40:21.06 entering replicative senescence,
00:40:24.11 and a great increase in the
00:40:26.06 number of beta-galactosidase positive cells.
00:40:30.23 All of this tells us that lamin B1
00:40:32.21 in some way, or the ratio of lamin B1 relative to lamin B2
00:40:37.16 and the A type lamins A and C,
00:40:39.19 is critically important in both regulating cell growth,
00:40:43.13 cell proliferation,
00:40:44.28 and in fact
00:40:48.07 entry into replicative senescence.
00:40:52.09 And we are now studying the mechanisms
00:40:54.24 by which lamin B1
00:40:57.17 acts in this fashion,
00:40:59.09 and the preliminary evidence indicates
00:41:01.27 that it's very important in regulating genes
00:41:04.10 involved in oxidative stress.
00:41:09.14 In closing, I would like to acknowledge
00:41:11.21 and thank all of the members of my own laboratory,
00:41:16.02 who have worked with us
00:41:18.09 in the recent past
00:41:20.14 and are presently in the lab,
00:41:23.01 as well as our collaborators
00:41:24.14 from here and overseas.
00:41:26.20 And I'd also like to thank our funding agencies,
00:41:30.16 the National Institute for General Medical Sciences,
00:41:33.15 the National Cancer Institute,
00:41:35.08 the Ellison Medical Foundation,
00:41:37.05 the Progeria Research Foundation,
00:41:39.08 and the Grus-Lipper Foundation.
00:41:41.20 Thank you.

Videos in this Talk
  • Part 1: Cytoskeletal Intermediate Filaments
    Part 1: Cytoskeletal Intermediate Filaments
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
  • Part 2: Nuclear Lamins
    Part 2: Nuclear Lamins
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Robert Goldman
Total Duration: 1:18:16
Recorded: July 2013
All Talks in Cell Biology

Talk Overview

In Part 1 of his talk, Dr. Goldman introduces us to cytoskeletal intermediate filaments beginning with an overview of IF formation and properties. Goldman focuses on vimentin to demonstrate that the assembly and disassembly of IFs are critical to cell shape change, lamellipodia formation and cell motility. Further experiments show that IF assembly and disassembly are regulated by kinases and phosphatases acting in response to growth factors and other signals.

In the second part of his talk, Goldman focuses on the nuclear lamins-a family of IFs found in the nucleus. Lamins have many critical roles including being a major determinant of nuclear size and structure. They are also necessary for DNA synthesis and repair and transcription by RNA polymerase II. Mutations in the lamin A gene are responsible for an astounding number of human diseases including the devastating early aging syndrome Hutchison Gilford Progeria. Studies of the nuclei from progeria cells are leading to a better understanding of the role of lamins in this disease and hope for treatment in the future.

Speaker Bio

Robert Goldman

Robert Goldman

Bob Goldman is Professor and Chair of the Department of Cell and Molecular Biology at the Feinberg School of Medicine at Northwestern University. He also has been a summer investigator at the Marine Biological Laboratory (MBL) in Woods Hole, since 1977. Goldman received his Ph.D. in biology from Princeton University. He was a postdoctoral fellow… Continue Reading

Playlist: Cytoskeletal Proteins

Related Resources

Cytoskeletal Intermediate Filaments Primary Papers

Guo, M., et al. (2013) The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics. Biophys. J. 105: 1563-1568.

Mahammad, S., et al. (2013) Giant Axonal Neuropathy-Associated Gigaxonin Mutations Impair Intermediate Filament Protein Degradation. J Clin Investigation. 123: 1964-1975.

Helfand, B et al (2011) Vimentin organization modulates the formation of lamellipodia. MBOC 22: 1274-1289.

Mendez, et al (2010) Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J 24: 1838-1851.

Helfand, B et al (2002) A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization. J Cell Biol. 157: 795-806.

Prahlad, V et al (1998) Rapid Movements of Vimentin on Microtubule Tracks: Kinesin-dependent Assembly of Intermediate Filament Networks J Cell Biol. 143: 159-170.

Cytoskeletal Intermediate Filaments Reviews:
Chang, L et al (2004) Intermediate Filaments Mediate Cytoskeletal Crosstalk Nature Rev Molec Cell Biol. 5: 601-613.

Godsel, L. et al (2007) Intermediate filament assembly: dynamics to disease. Trends in Cell Biol Goldman, RD, et al (2008) Intermediate filaments: versatile building blocks of cell structure Curr Op Cell Biol 20: 2028-2034

Eriksson, J. E. et al. (2009) Introducing Intermediate Filaments: from Discovery to Disease. J Clin Investigation 119: 1673-1771.

Herrmann, H. et al (2009) Intermediate filaments: primary determinants of cell architecture and plasticity. J Clin Investigation 119: 1772-1783.

Le´pinoux-Chambaud, C and Eyer, J. (2013) Review on intermediate filaments of the nervous system and their pathological alterations Histochem Cell Biol 140:13–22.

Chung, B-M, et al (2013) Networking galore: intermediate filaments and cell migration. Curr Op Cell Biol 25: 600-612.

The Nuclear Lamins Primary Papers:
Shimi, T, et al (2011) The Role of Nuclear Lamin B1 in Cell Proliferation and Senescence. Genes and Development 25:2579-2593.

Shimi, T et al (2008) The A- and B- Type Nuclear Lamin Networks: Microdomains Involved in Chromatin Organization and Transcription. Genes and Development 22: 3409-3421.

Goldman, RD, et al (2004) Accumulation of Mutant Lamin A Causes Progressive Changes in Nuclear Architecture in Hutchinson-Gilford Progeria Syndrome. PNAS 101: 8963-8968.

Spann, T et al (2002)Alteration of nuclear lamin organization inhibits RNA polymerase II–dependent transcription J Cell Biol 156:603-608.

The Nuclear Lamins Reviews:
Dechat, T, et al (2008) Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes and Development 22: 832-853

Butin-Israeli, V. et al (2012) Nuclear Lamin Functions and Disease. Trends in Genetics 28: 464-471.

Burke, B. and Stewart, CL (2013) The Nuclear Lamins: Felxibility in Function. Nat Rev Mol Cell Biol 14: 13-24.

 

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