Phosphorylation of keratin intermediate filaments ( IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells ( AEC) and that this disassembly is regulated by protein kinase C delta-mediated phosphorylation of keratin 8 ( K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C ( PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC delta and that the PKC delta peptide antagonist, delta V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

How timely transport of chemical signals between the distal end of long axonal processes and the cell bodies of neurons occurs is an interesting and unresolved issue. Recently, Perlson et al. presented evidence that cleavage products of newly synthesized vimentin, an intermediate filament (IF) protein, interact with mitogen-activated protein (MAP) kinases at sites of axon injury. These IF fragments appear to be required for the transport of these kinases to the cell body along microtubule tracks. The truncated vimentin is instrumental in signal propagation as it provides a scaffold that brings together activated MAP kinases (such as Erk 1 and Erk2), as well as importin beta and cytoplasmic dynein. The authors propose that this all-in-one transport complex has the extraordinary ability to travel towards the cell body and enter the nucleus where the kinases activate and influence gene expression so that a neuron can generate a timely response to injury.

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the 32P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

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.

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Until recently, the dynamic properties of intermediate filaments (IF) were attributed primarily to the exchange of subunits between a disassembled pool and polymerized 10nm filaments. During interphase, this subunit exchange process was thought to produce local modifications in IF structure. During cell division, shifts in the equilibrium between subunits and polymers were thought to lead to either the global or regional disassembly of IF networks, thereby facilitating their distribution into daughter cells. Recently, novel structural forms of IF that undergo rapid and directed transport in several cell types were revealed. Time-lapse observations of motile IF structures in different cell systems have also revealed novel insights into the mechanisms underlying the transport of cytoskeletal components throughout the cytoplasm and the molecular basis of the ‘crosstalk’ between different cytoskeletal systems.

In most vertebrate cells, intermediate filaments (IF) form a continuous structural network extending from the nuclear surface to the cell periphery. Their unique viscoelastic properties render them more resistant than either microtubules or microfilaments to deformation and other external physical

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH2-terminal domain of vimentin and the vimentin binding site at residues 143–188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin–vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.

Intermediate filaments (IF) are major cytoskeletal proteins of animal cells. They are comprised of a large family of >50 gene products that are capable of polymerizing into ∼10 nm diameter IF. The rules for IF polymerization from these protein subunits are complex. Some can form IF from a single type of protein chain (homopolymers), while others can form IF only when they are combined with more than one type of protein chain (heteropolymers). Within the cytoplasm of cells, IF form elaborate and complex networks. These are usually concentrated in the perinuclear region and radiate throughout the cytoplasm, where they are frequently associated with the cell surface (1, 26). The extent of sequence homology, the pattern of cell type specific expression, and the similarity of intron positions of their genes are properties that have been used to classify IF proteins into six different types (1–3). The largest number of IF proteins are categorized as the type I and II keratins, which are expressed mainly in epithelial cells. The keratins are obligatory copolymers that require both a type I and a type II protein to form IF. The expression of specific keratin pairs is precisely regulated during development, and this unique feature has led to their use as differentiation markers for epithelial cells. Vimentin, desmin, glial fibrillary acidic protein (GFAP), and peripherin are the four known type III IF proteins. Each of these type III proteins is capable of assembling into homopolymer IF. Among them, vimentin is unique in that it often forms a scaffold IF network before the expression and assembly of differentiation-specific IF proteins such as desmin, GFAP, and peripherin. Type IV IF proteins consist of the three neurofilament subunit proteins, NF-L, NF-M, and NF-H, as well as α-intenexin. Neurofilaments are obligatory heteropolymers, while α-internexin is self-assembly competent (4). Although the majority of IF proteins are expressed in the cytoplasm, the type V IF proteins (the nuclear lamins) are localized in the nucleus where they polymerize to form the nuclear lamina. The lamina is located at the interface between chromatin and the inner face of the nuclear envelope membrane. Recent observations demonstrate that the nuclear lamina is not only essential for the structural integrity of the nuclear envelope but is also involved in the process of DNA replication during S phase of the cell cycle (5). The type VI IF protein nestin has not been characterized extensively. It was discovered as an early developmental marker of neuroepithelial cells (3), which cannot form IF on its own but is capable of coassembling with vimentin to form IF (28). The IF proteins possess both well-conserved and nonconserved domains. The most conserved region is the central α-helical rod domain consisting of ∼310 amino acids, known to form coiled-coils in assembled IF. This central rod is flanked at both ends by NH2- and carboxyl-terminal non-α-helical regions that are not as well conserved among the various types of IF proteins. It has often been speculated that these less conserved end domains may be involved in the cell type specific functions of IF, as well as their higher order structure. Within the rod domain, two stretches of sequence are highly conserved that are known to play important roles in IF assembly (1). They are located at the beginning (the IA region) and at the end (the 2B region) of the rod. Although the details of IF structure remain to be determined, the most widely accepted model is that IF are comprised of four protofibrils. Individual protofibrils contain two protofilaments, each of which is constructed from linear arrays of antiparallel and staggered dimers. The basic building block is the dimer, consisting of two parallel and in register protein chains (6). IF are known to provide mechanical integrity to cells. In vitro they exhibit unusual viscoelastic properties making them much more resistant to mechanical stress when compared with microtubules and actin-containing microfilaments (7). Their mechanical role has been demonstrated at the tissue level in vivo by the finding that numerous human blistering diseases are caused by point mutations in epidermal keratin genes (8). This is also supported by studies in which targeted expression of dominant negative keratin mutations in mice results in severe blistering of the skin. The latter lesions appear to be caused by the weakening of the mechanical properties of keratin IF in epidermal keratinocytes, with concomitant loss of cell shape and cell lysis (8). In the case of type III IF, it has been shown in mice that ablation of the desmin gene leads to the degeneration of cardiac and skeletal muscle, suggesting that desmin plays an important role in maintaining the mechanical integrity of muscle cells (9). In a similar fashion, defective expression of neurofilaments in animal models results in the aberrant organization of neurofilament networks, which is sufficient to generate phenotypes reminiscent of neurological diseases (10). IF are closely associated with various types of junctional complexes at cell surfaces, including desmosomes and hemidesmosomes in epidermal cells (11) and focal adhesions in fibroblasts (12). These associations are mediated by a growing family of proteins known as intermediate filament associated proteins (IFAPS; ref 13). These include plectin, desmoplakin, and BPAG1n/dystonin (13). IFAPs have also been shown to play an important role in the interactions among cytoskeletal systems. For example, plectin (14) BPAG1n/dystonin (15) and fimbrin (32) possess both IF and actin binding sites (14, 15). The importance of IFAPs in cytoskeletal integrity has been highlighted by studies of BPAG 1-null mice and patients afflicted with muscular dystrophy associated with epidermolysis bullosa simplex (MD-EBS). In the case of MD-EBS, the effects are linked to the expression of truncated plectin molecules (14, 16). Based on their biochemical properties in vitro and their known roles in contributing to the mechanical properties of cells, many researchers have assumed that IF are static elements of a cell's cytoskeletal repertoire. However, it has become increasingly obvious over the past decade that IF are dynamic structures. For example, dramatic changes in IF network organization occur in mitotic cells and in spreading cells after cytokinesis or, in the case of cultured cells, after trypsinization and replating (25). In addition, the nuclear lamina is dismantled and dispersed throughout the cytoplasm during mitosis and is reassembled during the reformation of daughter cell nuclei (5). Studies designed to determine the properties of IF during interphase have been carried out using microinjection techniques. Experiments involving the microinjection of soluble biotinylated vimentin and type I keratin subunits have revealed that endogenous polymerized IF can incorporate exogenous subunits (17, 18). However, if the amount of micro-injected type I keratin is above a certain concentration, there is a rapid induction of the disassembly of endogenous keratin tonofibrils. Presumably, this is because of the formation of types I and II heterodimers, which are required for keratin IF polymerization. Based on these findings, we have speculated that an excess of type I protein could effectively remove type II from an exchangeable pool of subunits, thereby displacing additional subunit proteins from keratin IF, ultimately resulting in their disassembly (17). These microinjection results suggest that there is an equilibrium state between exchangeable subunits and polymerized IF. By far the most convincing evidence supporting the dynamic properties of IF in vivo has been derived from fluorescence recovery after photobleaching (FRAP) experiments. These experiments on IF in living cells were initially carried out following the microinjection of rhodamine-conjugated vimentin (19). The results show that fluorescent vimentin fibrils are capable of recovering their fluorescence after photobleaching. This fluorescence recovery appears to be uniform all along the bleach zones, indicating that in vivo IF are apolar filaments. Unfortunately, in these studies only the initial phases of recovery could be monitored because of the low level of fluorescence emission of the rhodamine-tagged vimentin fibrils. This made it impossible to measure the t1/2 for full recovery (19). More recently, we have been able to track total recovery of fluorescence in bleach zones across vimentin fibrils tagged with green fluorescent protein (GFP)-vimentin (see below; refs 20, 21). To further our understanding of the properties of IF in vivo, we found it essential to develop a technique that would allow us to study IF for prolonged time periods. This has involved the use of GFP-tagged vimentin. Live cells transfected with GFP-vimentin contain IF networks that are indistinguishable from fixed/stained cells studied by immunofluorescence. Furthermore, we have found that these transfected cells can be observed for intervals of more than 1 h (21). The results of our initial studies using GFP-vimentin reveal that the constituents of IF networks are much more dynamic than previously thought. This has been shown by time-lapse confocal microscopy, which demonstrates that individual vimentin fibrils alter their shape, change their length, and either appear or disappear in various regions of the cytoplasm. IF shorten at mean rates of 0.42 µM/min and elongate or extend at average rates of 0.4 µM/min (Fig. 1a–f). Frequently, neighboring vimentin fibrils translocate at different rates and sometimes in opposite directions. This is indicated by the different rates of movements of photobleach marks that average 0.24 µm/min (Fig. 1g–i; ref 21). When GFP-vimentin expressing cells are treated with nocodazole, the average rate of these movements is reduced by 60%, and the same is true of cytochalasin B. These results indicate that the translocation of IF is somewhat dependent on both microtubules and microfilaments (21). Our measured rates of IF bleach zone translocation compare favorably with those that have been measured for microtubules and microfilaments using photobleaching and photoactivation, although the underlying mechanisms are probably different (e.g., treadmilling for microtubules and microfilaments). These techniques have revealed that the rates for translocation along kinetochore microtubules range from ∼0.12 to 0.7 µm/min (22, 23). In the case of actin-containing stress fibers, average rates of 0.29 µm/min have been recorded (24). As for actin-containing fibrils in la-mellipodia, a faster speed of 0.79 µm/min has been reported (27). In addition to the above, rapid movements of short vimentin IF, termed 'squiggles', are observed. These are seen primarily in the peripheral regions of the cytoplasm where they translocate at an average rate of 3.3 ± 1.9 µm/min with speeds ranging from 1.3–11.4 µm/min (Fig. 1j–l). These latter movements are almost completely inhibited when microtubules are disassembled with nocodazole (21), indicating the involvement of microtubule-based motors (see below). The opportunity to study individual vimentin fibrils in cells transfected with GFP-vimentin for longer time intervals in vivo has also permitted us to calculate the t1/2 for full recovery of fluorescence after photobleaching. FRAP analyses of GFP-vimentin fibrils yield a t1/2 = 5 ± 3 min. This rate is similar to those obtained for microtubules and microfilaments in vivo (21), indicating that the speeds of subunit/polymer exchange for all three cytoskeletal elements are comparable. The assembly of IF in vitro has been studied extensively, and the accumulated data suggest that the process requires no energy and no accessory proteins. However, details of the in vivo assembly process remain largely unknown. To begin to investigate this process, we have monitored the behavior of GFP-vimentin by time-lapse confocal microscopy in spreading BHK-21 cells following trypsinization and replating. During the first 30–45 min of spreading, the majority of the GFP-vimentin is localized as a filamentous aggregate in the perinuclear area (25). However, in the peripheral regions of the cell, most of the GFP-vimentin is present as nonfilamentous vimentin particles (also called 'dots' (20); Fig. 2a, b). Within 1–1.5 h after replating, the vimentin particles in the periphery appear to be transformed into the short fibrous squiggles (see above; ref 21). After 3 h, as cells spread even further, the number of particles and squiggles decreases, apparently replaced by the long vimentin fibrils that typify the IF networks of spread cells. From these time-lapse observations, it appears that the assembly of the vimentin IF network involves three morphologically distinct steps: non-filamentous particles, short fibrous squiggles, and long fibrils (20). Time-lapse observations have also provided us with remarkable new insights into the behavior of the vimentin particles. At any given time during the early stages of cell spreading, these particles can be seen to undergo either vibrational movements with no net translocation or rapid and unidirectional movements. The same particle can switch back and forth between these two types of movements. Time-lapse measurements of these rapidly moving particles give an average of 0.55 ± 0.24 µm/s with peak velocities of 1 µm/s. The movement of the particles is along relatively straight paths primarily oriented toward the peripheral regions of the cell. In a few cases, vimentin particles move away from the cell surface toward the nucleus (Fig. 2c–e). Furthermore, the rapid translocation is abolished after the treatment of cells with nocodazole, suggesting that particle motility depends on microtubules. Taken together, these observations suggest the involvement of a plus-end directed microtubule-based motor in the transport of nonfilamentous vimentin from the perinuclear to the peripheral region of the cell. Indeed, immunofluorescence observations using antibodies specific for conventional kinesin indicate that the majority of vimentin particles in the peripheral regions of the cell colocalize with conventional kinesin (20). Overall, our observations of GFP-vimentin in live spreading cells suggest that IF assembly in vivo is a dynamic process involving kinesin, microtubules, and an energy source such as ATP (20, 21). From the above described studies, it can be inferred that the organizational state of IF in vivo is regulated by an equilibrium between IF subunits and polymerized IF. This led us to the development of specific inhibitors of IF polymerization for use in studies of their function in vivo (26). The inhibitors are mimetic peptides corresponding to the conserved amino acid sequence of the helix 1A regions of IF proteins that are known to be potent and specific disruptors of IF assembly in vitro. On microinjection into fibroblasts, these peptides induce rapid disruption of IF networks, followed by the disassembly of both microtubule and microfilament networks, as the cells alter their shape from a flattened fibroblast configuration to a rounded morphology (Fig. 2f–i). The underlying reason for these dramatic effects is not clear. However, it is possible that IF, together with IFAPs, play pivotal roles in linking the three cytoskeletal elements into interdependent functional units. Therefore, it is likely that the disruption of IF networks alters the functional states of IFAPs, which in turn leads to the profound alterations in the organization of all three cytoskeletal systems that is seen following the injection of the 1A peptide (13, 26). These observations also lend further support to the role of IF in maintaining the integrity and the mechanical properties of the cytoplasm. The results from this laboratory as well as the work of others demonstrate that IF are dynamic structures in living cells. Their dynamic properties are regulated by complex and largely unknown mechanisms involving linkages to other cytoskeletal elements, such as microtubules and microfilaments, and with molecular motors, such as kinesin. It appears likely that protein phosphorylation plays a role in the subunit/polymer exchange process underlying many of their dynamic properties. For example, global and localized phosphorylation of IF proteins has been correlated with the dramatic changes in their organizational and assembly states in mitotic cells (29) and in the regional disassembly of IF networks in cleavage furrows (30). Furthermore, during interphase, it has been suggested that the state of IF assembly is regulated by a balance between the activities of protein kinases and phosphatases (31). Clearly, the properties of IF suggest that they play important roles in many normal physiological activities ranging from cellular mechanics to signal transduction (13). This work has been supported by a MERIT award from the National Institute of General Medical Sciences.

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.

Recent research highlights the roles of cytoskeletal intermediate filaments (IFs) and their interactions with both the cell surface and other cytoskeletal systems in maintaining cellular integrity and the mechanical properties of cytoplasm. This has been demonstrated by analyses of mutations in IF-associated proteins (IFAPs) that are involved in connecting IFs to cell surface junctions. New data also point to the role of IFAPs as molecular 'nuts and bolts' in the construction of an integrated cytoplasmic architecture. This is highlighted by the initial descriptions of a family of multifunctional molecules that are capable of bridging IFs to other cytoskeletal elements. These findings, together with the development of specific peptide inhibitors capable of disassembling IF networks in vivo, are paving the way to the identification of new cellular functions for IFs and IFAPs.

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.

During mitosis in BHK-21 baby hamster kidney cells the hyperphosphorylation of the type III intermediate filament (IF) protein vimentin is accompanied by the disruption of the IF network into punctate, protofilamentous structures. In this study, the morphological and biochemical changes of IFAP 300, a 300-kDa IF-crossbridging protein, are examined during mitosis. Double-label immunofluorescence shows that the distribution of IFAP 300 coincides with the typical filamentous pattern displayed by vimentin in interphase cells, whereas in mitotic cells it is reorganized into a punctate, nonfilamentous pattern. Accompanying these latter morphological changes, IFAP 300 is phosphorylated at a unique, mitosis-specific site. Comparison of the sites phosphorylated in cultured cells with those phosphorylated in vitro by various kinases suggests that IFAP 300 is phosphorylated by the same two kinases that phosphorylate vimentin during mitosis. One of these is p34cdc2 protein kinase, which appears to be responsible for the phosphorylation of the mitosis-specific site. The other kinase phosphorylates IFAP 300 in vitro at a site that is also found in the protein immunoprecipitated from either mitotic or interphase cells. In contrast to vimentin, the phosphorylation levels of IFAP 300 are not obviously altered between interphase and mitosis. Our results show that IFAP 300 is a physiological substrate for p34cdc2 and that this kinase may be involved in the mitotic reorganization of IFAP 300 by phosphorylating a mitosis-specific site. Taken together with our previous results, this study suggests that the activation of p34cdc2 coordinates the mitotic reorganization of the vimentin IF network both by severing IF-IF connections mediated by IFAP 300 and by disassembling individual IFs into protofilaments.