Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

Alteration of vimentin intermediate filament expression during differentiation of human hemopoietic cells

We describe the alterations of vimentin intermediate filament (IF) expression in human hemopoietic committed precursors as they differentiate into mature cells of the erythroid, granulomonocytic, megacaryocytic and lymphoid lineages. A double labelling fluorescence procedure was used to identify hemopoietic cells expressing lineage-specific antigens and to decorate the vimentin IF network. Whereas very early progenitors from each lineage expressed vimentin, the density and organization of the network differed strikingly as the cells matured on a given pathway. T lymphocytes, monocytes and granulocytes retained vimentin expression at all stages of maturation. In contrast, megakaryoblasts lose vimentin expression at a very early stage of differentiation, erythroblasts at variable steps between the committed erythroid cell and the red cell. Finally, B lymphocytes tend to lose vimentin expression later when they mature into plasma cells.     

A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton

The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.     

Specific types of prosomes are associated to subnetworks of the intermediate filaments in PtK1 cells.

Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)-- fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.     

Sorting out IF networks: consequences of domain swapping on IF recognition and assembly

Vimentin and keratin are coexpressed in many cells, but they segregate into two distinct intermediate filament (IF) networks. To understand the molecular basis for the sorting out of these IF subunits, we genetically engineered cDNAs encoding hybrid IF proteins composed of part vimentin and part type I keratin. When these cDNAs were transiently expressed in cells containing vimentin, keratin, or both IFs, the hybrid IF proteins all recognized one or the other or both networks. The ability to distinguish networks was dependent upon which segments of IF proteins were present in each construct. Constructs containing sequences encoding either helix 1B or helix 2B seemed to be the most critical in conferring IF recognition. At least for type I keratins, recognition was exerted at the level of dimer formation with wild-type type II keratin, as demonstrated by anion exchange chromatography. Interestingly, despite the fact that swapping of helical domains was not as deleterious to IF structure/function as deletion of helical domains, keratin/vimentin hybrids still caused structural aberrations in one or more of the cytoplasmic IF network. Thus, sequence diversity among IF proteins seems to influence not only coiled-coil but also higher ordered associations leading to 10-nm filament formation and/or IF interactions with other cellular organelles/proteins.     

Fast repetition rate (FRR) fluorometry: variability of chlorophyll a fluorescence yields in colonies of the corals, Montastraea faveolata (w.) and Diploria labyrinthiformes (h.) recovering from bleaching

Recently, an underwater version of a fast repetition rate fluorometer (FRRF) was developed for the non-destructive study of fluorescence yields in benthic photoautotrophs. We used an FRRF to study bleached colonies of the corals, Montastraea faveolata and Diploria labyrinthiformes at sites surrounding Lee Stocking Island, Exuma, Bahamas, to assess their recovery from bleaching ( approximately 1 year after the initial bleaching event) induced by elevated temperatures. The steady state quantum yields of chlorophyll a fluorescence (DeltaF'/F'(m)) from photosystem II (PSII) within coral colonies were separated into three categories representing visibly distinct degrees of bleaching ranging from no bleaching to completely bleached areas. Differences in DeltaF'/F'(m) were significantly different from bleached to unbleached regions within colonies. Dark, unbleached regions within colonies exhibited significantly higher DeltaF'/F'(m) values (0.438+/-0.019; mean+/-S.D.) when compared to lighter regions, and occupied a majority of the colonies' surface area (46-73%). Bleached regions exhibited significantly lower DeltaF'/F'(m) (0.337+/-0.014) and covered only 7-25% of the colonies' surface area. The observations from this study suggest that zooxanthellae in bleached regions of a colony exhibit reduced photosynthetic activity as long as one year after a bleaching event and that in situ fluorescence techniques such as FRRF are an effective means of studying coral responses and recovery from natural or anthropogenic stress in a non-destructive manner.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching

One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 micron wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 micron diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assembly-disassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33 degrees C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the half-spindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.     

Keeping the Vimentin Network under Control: Cell–Matrix Adhesion–associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts

Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors, which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.     

Withaferin a alters intermediate filament organization, cell shape and behavior

Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.     

Integrity of intermediate filaments is associated with the development of acquired thermotolerance in 9L rat brain tumor cells

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament-targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45°C for 15 min (priming heat-shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat-shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent-extractability studies. In cells treated with WA before the priming heat-shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat-shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat-shock only. HSC70 was found to be co-localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.     

Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.     

Modulation of vimentin containing intermediate filament distribution and phosphorylation in living fibroblasts by the cAMP-dependent protein kinase

Microinjection of the purified catalytic subunit of the cAMP-dependent protein kinase (A-kinase) into living rat embryo fibroblasts leads to dramatic changes in vimentin intermediate filament (IF) organization, involving the collapse of the filaments into tight bundles. In some cell types, this rearrangement of the IF proceeds further, leading to an apparent loss of filament integrity, resulting in a punctate staining pattern throughout the cytoplasm. Both these types of IF rearrangement are fully reversible, and similar to structural changes previously described for IF during mitosis. As shown by electron microscopy, in rat embryo fibroblasts these changes in IF structure do not involve the loss of the 10-nM filament structure but instead correspond to the bundling together of 25 or more individual filaments. Metabolic pulse labeling of injected cells reveals that accompanying these changes in IF organization is a dramatic increase in vimentin phosphorylation which appears maximal when the IF are fully rearranged. However, this increase in IF phosphorylation is not accompanied by any significant increase in soluble vimentin. Analysis of the sites of phosphorylation on vimentin from injected cells by either V8 protease cleavage, or two-dimensional tryptic peptide mapping, revealed increased de novo phosphorylation of two vimentin phosphopeptides after microinjection of A-kinase. These data strongly suggest that the site-specific phosphorylation of vimentin by A-kinase is responsible for the dynamic changes in IF organization observed after injection of the kinase into living cells, and may be involved in similar rearrangement of the IF previously described during mitosis or after heat shock.     

Protein kinase C associates with intermediate filaments and stress fibers.

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J. Biol. Chem. 262, 2291-2297; Mochly-Rosen, D., et al. 1987 Proc. Natl. Acad. Sci. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the beta-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin+ and vimentin- clones of SW13 (a human adrenal carcinoma cell line), CEM (a human T cell line), U937 (a histiocytic myeloid cell line), and HL60 (a promyelocytic leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the beta-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentin- mutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers.     

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.     

Microinjection of monoclonal antibodies to vimentin, desmin, and GFA in cells which contain more than one IF type

Microinjection of antibodies to vimentin into fibroblast cell lines causes intermediate filaments (IFs) to build perinuclear caps. We have extended these findings by microinjection of monoclonal antibodies specific for different IF types to non-epithelial cell lines of human origin, which co-express two different IF proteins. Thus GFA and vimentin IgGs have been microinjected in separate experiments into a glioma cell line, desmin and vimentin IgGs into RD cells, and vimentin IgGs into a cell line which co-expresses neurofilaments and vimentin. In all instances, microinjection of a single antibody causes the formation of perinuclear caps in which the two different IF proteins co-localize, suggesting that vimentin and the second IF type present in each cell line localize to the same 10-nm filaments. Immunoelectron microscopy using desmin and vimentin antibodies made in different species and appropriate second antibodies labelled with 5 and 20 nm gold particles confirm this result for RD cells. When Fab' fragments of the vimentin IgGs are microinjected into different cell types, formation of perinuclear caps is observed in immunofluorescence microscopy. In RD cells immunoelectron microscopy shows that the Fab' fragments induce caps which appear less dense than the caps seen after microinjection of IgGs.     

A novel type of regulation of the vimentin intermediate filament cytoskeleton by a Golgi protein

Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877-894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.