Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Transient increase in vimentin phosphorylation and vimentin-HSC70 association in 9L rat brain tumor cells experiencing heat-shock

Characteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45°C. During heat-shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher-order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat-shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat-shock protein70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co-insolubilized during the heat-shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat-shock genes.     

Intermediate vimentin filaments and their role in intracellular organelle distribution

Intermediate filaments (IF) represent one of three main cytoskeletal structures in most animal cells. The human IF protein family includes about 70 members divided into five main groups. The characteristic feature of IF is that in various cells and tissues they are formed by proteins of different groups. Structures of all IF proteins follow a unique scheme: a central α-helical part is flanked at the N and C ends by positively charged polypeptide chains devoid of a clear secondary structure. The central part is highly conserved for all proteins in all animals, whereas the N and C termini strongly differ both in size and amino acid composition. This review covers the broad spectrum of recent investigations of IF structure and diverse functions. Special attention is paid to the regulatory mechanisms of IF functions, mainly to phosphorylation by different protein kinases whose role is well studied. The review gives examples of hereditary diseases associated with mutations of some IF proteins, which point to an important physiological role of these cytoskeletal structures.     

Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38^MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38^MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38^MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments. J. Cell. Biochem. 71:169–181, 1998. © 1998 Wiley-Liss, Inc.     

Engagement of vimentin intermediate filaments in hypotonic stress

Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano-responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo-osmotic–induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo-osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin-1 (KIF5B) block the hypo-osmotic shock–induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin-dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo-osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.     

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.     

Induction of vimentin modification and vimentin-HSP72 association by withangulatin A in 9L rat brain tumor cells

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 μM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 μM and a completely upshift mobility was observed after 5 h treatment at 50 μM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.     

A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization

We present evidence that vimentin intermediate filament (IF) motility in vivo is associated with cytoplasmic dynein. Immunofluorescence reveals that subunits of dynein and dynactin are associated with all structural forms of vimentin in baby hamster kidney-21 cells. This relationship is also supported by the presence of numerous components of dynein and dynactin in IF-enriched cytoskeletal preparations. Overexpression of dynamitin biases IF motility toward the cell surface, leading to a perinuclear clearance of IFs and their redistribution to the cell surface. IF-enriched cytoskeletal preparations from dynamitin-overexpressing cells contain decreased amounts of dynein, actin-related protein-1, and p150Glued relative to controls. In contrast, the amount of dynamitin is unaltered in these preparations, indicating that it is involved in linking vimentin cargo to dynactin. The results demonstrate that dynein and dynactin are required for the normal organization of vimentin IF networks in vivo. These results together with those of previous studies also suggest that a balance among the microtubule (MT) minus and plus end-directed motors, cytoplasmic dynein, and kinesin are required for the assembly and maintenance of type III IF networks in interphase cells. Furthermore, these motors are to a large extent responsible for the long recognized relationships between vimentin IFs and MTs.     

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.     

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.     

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.     

Modeling alpha-helical coiled-coil interactions: the axial and azimuthal alignment of 1B segments from vimentin intermediate filaments

Attempts at predicting the relative axial alignments of fibrous protein molecules in filamentous structures have relied upon representing the (multichain) molecular structure by a one-dimensional sequence of amino acids. Potential intermolecular ionic and apolar interactions were counted and determined as a function of the relative axial stagger between the molecules. No attempts were made to consider the azimuthal aspect of the interacting molecules and neither were apolar or ionic energy terms used. Surprisingly, this simple approach proved remarkably informative and yielded accurate predictions of the axial periods present. However, a more comprehensive analysis involving the energetics of aggregation taking due regard for the relative azimuths of the molecules as well as their separation should decrease the noise level in the calculations and reveal other pertinent information. Toward that end, we have modeled the interaction between two alpha-helical coiled-coil segments in intermediate filament molecules (1B segments from human vimentin). The relative axial alignment and polarity of the molecules is already known from detailed crosslinking studies and this provides a criterion against which the success (or otherwise) of the modeling can be judged. The results confirm that an antiparallel alignment of two 1B segments is preferred over any of the parallel options (as observed experimentally). The calculated axial alignment, however, is not identical to that observed from detailed crosslinking studies indicating that other parts of the molecule (probably the head and tail domains as well as other coiled-coil segments) have a crucial role in determining the precise mode of axial aggregation. The results also show that the apolar interactions seem to be significantly less important in the alignment process than the ionic ones. This is consistent with the observation of a well-defined period in the linear disposition of the charged (but not apolar) residues along the length of the outer surface of the vimentin molecule.     

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: Evidence from gene knockout studies

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.     

Sites of actin filament initiation and reorganization in cold-treated tobacco cells

Cytoskeletal proteins assemble into dynamic polymers that play many roles in nuclear and cell division, signal transduction, and determination of cell shape and polarity. The distribution and dynamics of microtubules (MTs) and actin filaments (AFs) are determined, among other factors, by the location of their nucleation sites. Whereas the sites of microtubule nucleation in plants are known to be located under the plasma membrane and on the nuclear envelope during interphase, there is a striking lack of information about nucleation sites of AFs. In the studies reported herein, low temperature (0 °C) was used to de‐polymerize AFs and MTs in tobacco BY‐2 (Nicotiana tabacum L.) cells at interphase. The extent of de‐polymerization of cytoskeletal filaments in interphase cells during cold treatment and the subcellular distribution of nucleation sites during subsequent recovery at 25 °C were monitored by means of fluorescence microscopy. The results show that AFs re‐polymerized rapidly from sites located in the cortical region and on the nuclear envelope, similarly to the initiation sites of MTs. In contrast to MTs, however, complete reconstitution of AFs was preceded by the formation of transient actin structures including actin dots, rods, and filaments with a dotted signal. Immunoblotting of soluble and sedimentable protein fractions showed no changes in the relative amounts of free and membrane‐bound actin or tubulin.     

Cytoskeleton of human mononuclear cells as a possible peripheral marker for phenylalanine neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of ³²P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the ³²P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the ³²P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.     

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and—in some cases—fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.     

Dissecting the contribution of actin and vimentin intermediate filaments to mechanical phenotype of suspended cells using high-throughput deformability measurements and computational modeling

Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement.