Vimentin on the move: new developments in cell migration

The vimentin gene ( VIM) encodes one of the 71 human intermediate filament (IF) proteins, which are the building blocks of highly ordered, dynamic, and cell type-specific fiber networks. Vimentin is a multi-functional 466 amino acid protein with a high degree of evolutionary conservation among vertebrates. Vim −/− mice, though viable, exhibit systemic defects related to development and wound repair, which may have implications for understanding human disease pathogenesis. Vimentin IFs are required for the plasticity of mesenchymal cells under normal physiological conditions and for the migration of cancer cells that have undergone epithelial–mesenchymal transition. Although it was observed years ago that vimentin promotes cell migration, the molecular mechanisms were not completely understood. Recent advances in microscopic techniques, combined with computational image analysis, have helped illuminate vimentin dynamics and function in migrating cells on a precise scale. This review includes a brief historical account of early studies that unveiled vimentin as a unique component of the cell cytoskeleton followed by an overview of the physiological vimentin functions documented in studies on Vim −/− mice. The primary focus of the discussion is on novel mechanisms related to how vimentin coordinates cell migration. The current hypothesis is that vimentin promotes cell migration by integrating mechanical input from the environment and modulating the dynamics of microtubules and the actomyosin network. These new findings undoubtedly will open up multiple avenues to study the broader function of vimentin and other IF proteins in cell biology and will lead to critical insights into the relevance of different vimentin levels for the invasive behaviors of metastatic cancer cells.


Introduction
Vimentin is an intermediate filament (IF) protein whose name is derived from the Latin word vimentum, which means wickerwork 1 . Early observations with immunofluorescence microscopy revealed a complex fiber network, distinct from the already-known keratin system in the cytoskeleton of epithelial cells 1 . In mouse development, vimentin initially emerges in a highly migratory cell type (that is, when the embryo is still a two-layered epithelium and ectodermal cells start to migrate into the newly forming "mesodermal cleft"). In these first mesenchymal cells, keratin genes are turned off and the vimentin gene is turned on 2 . Postnatal expression of vimentin is restricted to fibroblasts, endothelial cells, lymphocytes, and several specialized cells of the thymus and the brain 3,4 . Moreover, it was observed early on that vimentin is significantly expressed in most cell types, particularly tumor cells, when the cells are taken from primary tissues and transitioned into culture 5 . The mechanism behind this widespread expression of vimentin is a serum response element in the VIM promoter, which responds to factors present in the serum that culture media are complemented with 6,7 . Therefore, many cell types expressing vimentin in culture are not ideal models to study the genuine biological functions of vimentin. However, with appropriate cell systems, it has been demonstrated that vimentin plays an important role in various physiological situations. For instance, upregulation of vimentin in cultured epithelial cells 8,9 and in vivo 10 correlates with epithelial-mesenchymal transition (EMT), a process that occurs during development, wound healing, and cancer metastasis 11 . Though originally described as a "skeletal" element of cells, the vimentin filament network was revealed by livecell imaging studies to be a very dynamic system 12 . Specifically, FRAP (fluorescence recovery after photobleaching) studies demonstrated that vimentin in interphase BHK-21 cells had a recovery half-time of 5 ± 3 minutes 12 , exhibiting dynamic properties similar to those of microtubules 13 and actin filaments 14 .
Small molecules for the selective targeting of vimentin (and other IFs) are currently not available, which has limited mechanistic understanding of this cytoskeletal component. The first global vimentin knockout mouse was generated 25 years ago and described as having no phenotype 15 , which was frequently and mistakenly taken as evidence that, despite its extreme evolutionary conservation in vertebrates 16

Novel roles of vimentin in cell migration
Vimentin promotes the migration of different cell types It is well appreciated that motile and invasive cells express higher levels of vimentin 47,48 and that vimentin knockout or knockdown attenuates the migration of fibroblasts 48,49 , leukocytes 20 , astrocytes 50 , and various cancer cell types 8,51,52 . For a broader overview of the functions of vimentin and other IFs in cell biology 53 (and cell migration in particular), we refer the readers to previous reviews 54-57 . Here, we specifically focus on the most recent studies illuminating how vimentin orchestrates cytoskeletal rearrangements and mechano-signaling to promote cell migration. In particular, we will discuss how the flexibility of the vimentin scaffold is modulated to provide a plastic "net" dynamically enforcing the rigid actomyosin motor system.
Vimentin filaments pattern microtubules during directed migration Establishment of persistent cell polarity is a key property of migrating cells responding to internal and external signals that guide directionality of movement 58 . The high turnover rate of the microtubule network, which occurs in the order of 3 to 5 minutes, stabilizes cell polarity during directed cell migration 59,60 . The vimentin filament network is closely associated with, and functionally dependent on, microtubules 61-63 and microtubule-associated molecular motors 64,65 . This is reflected in the drastic vimentin reorganization, often as an apparent "collapse" around the cell nucleus, upon disrupting microtubules with colchicine 62 . Recent work by Gan et al. used a systematic quantitative approach to characterize the co-dependent behavior of these two cytoskeletal systems during cell migration 66 . The authors subjected a retinal pigment epithelium (RPE) cell line expressing fluorescently tagged vimentin and tubulin (under the control of their endogenous promoters) to a scratch wound assay in a confluent monolayer followed by live-cell imaging and computational image analysis 66 . The study revealed that vimentin filaments are stable for up to 20 minutes after nocodazole treatment and that microtubules that associated with vimentin were more resistant to nocodazole treatment. Furthermore, microtubules used vimentin filaments as a growth template after nocodazole washout. The major conclusion from this work is that, during directed cell migration, long-lived vimentin filaments guide the growth of microtubule plus ends along the preceding microtubule tracks, thus providing a form of "memory" required for the continuous maintenance of cell polarity by the short-lived microtubules 66 . The molecular nature of the microtubule-vimentin interaction is not clear at present, although there is strong evidence that it is mediated via other proteins, such as adenomatous polyposis coli (APC) 67 , which links vimentin to microtubules, or it may be mediated via post-translational modifications (PTMs) 68 , such as phosphorylation. Additionally, it remains to be determined whether, and how, this mechanism applies to cells migrating in vivo, since RPE cells in vivo express keratins but lack vimentin 69 .

Vimentin regulates cell migration by restricting actin flow and aligning traction stress
Cell migration is dependent on actin filaments, which reorganize into different arrays to support the formation of membrane protrusions (for example, lamellipodia and filopodia) and propel the cell along its substrate 70 . Vimentin interacts with actin filaments directly via its tail domain 71 and indirectly via the cytolinker protein plectin 72 . Another vimentin binding partner, the capping protein (CP) regulator CARMIL2 (CP, Arp2/3, myosin-I linker 2), facilitates lamellipodia formation and cell migration in a vimentin-dependent manner 73 .
Jiu et al. showed recently that transverse arcs, which are actin bundles containing the motor protein myosin II, are essential for the retrograde flow of small vimentin particles and their incorporation into perinuclear vimentin filaments in the osteosarcoma U2OS cells 74 . The small vimentin particles, called squiggles 75 , represent intermediates of synthesis-independent filament turnover that occurs through severing and re-annealing 76 or subunit exchange 77 . Upon depletion of transverse arcs by knockdown of tropomyosin 4, which recruits myosin II 78 , retrograde flow and perinuclear localization of vimentin were lost 74 . While vimentin deficiency did not affect stress fiber formation in this particular system, it caused transverse arcs to pull away from the leading edge, suggesting that vimentin controls actin dynamics by restricting the retrograde flow of transverse arcs 74 . The cross-talk between vimentin and actin transverse arcs is dependent on plectin 74 and appears to be important for nuclear positioning, a key element of cell migration, and other processes 79 . To that end, vimentin was recently shown to interact with the nuclear pore complex protein Nup88 80 , which may bear consequences to nuclear positioning during cancer cell migration 81 .
Because vimentin filaments are highly dynamic, a key question is how the various physical states of the network control cellular behavior. For example, phosphorylation-dependent vimentin disassembly at the cell periphery is required for the formation of actin-based lamellipodia membrane protrusions 49 . The Danuser group developed a novel computational method to analyze vimentin filaments and showed that long (>4 µm) vimentin fibers serve as a load-bearing scaffold to buffer traction stress during single-cell migration 82 . Using traction force microscopy on non-immortalized human skin fibroblasts, the authors observed that actin moved 14 times faster in areas devoid of vimentin and two times faster in areas containing a rarefied vimentin "mesh" when compared with areas of the cell that were occupied by fibrous vimentin 82 . Thus, these findings align with the work by Jiu et al. 74 with respect to the function of filamentous vimentin in restraining retrograde actin flow. Additionally, traction forces distributed non-specifically throughout the interface between the cell and the substrate in vimentin-deficient cells while, in the presence of vimentin, actomyosin forces were redirected to peripheral adhesions 82 . Therefore, the current understanding is that mature vimentin fibers restrict the formation of lamellipodia and actin flow while facilitating the alignment of traction forces to promote single-cell migration in collaboration with microtubules ( Figure 1).

Vimentin promotes collective cell migration by restraining traction forces and supporting lateral cell-cell contacts
In addition to vimentin, actin transverse arcs regulate the perinuclear localization of nestin 74 , an IF protein that cannot form filaments on its own but can co-assemble with vimentin in various cell types, such as astrocytes 83 . Astrocytes are specialized glial cells critical for central nervous system (CNS) function 84 . The IF cytoskeleton of astrocytes is composed of vimentin, nestin, and glial fibrillary acidic protein (GFAP) 83,85 . Whereas GFAP is the major IF protein of mature astrocytes under basal conditions, vimentin is highly expressed by astrocytes during normal development and in CNS injury 85 . In developing Xenopus laevis embryos, vimentin-expressing cells first appear lining the forming neural tube 86 , indicating that these cells are radial glia guiding migratory neuronal cells 87 .
There is strong evidence that astrocyte migration is implicated in CNS development 88,89 , injury 90,91 , and glioma tumor formation 92 . Combined reduction of the protein levels of vimentin, GFAP, and nestin decreases astrocyte speed, directionality, and persistence of movement during collective cell migration 93 , the coordinated movement of cells as groups, in a manner dependent on cell-cell contact 94 . In a scratch wound assay using primary rodent astrocytes, knockdown of vimentin, along with GFAP and nestin, promotes an increase in actin stress fibers perpendicular to the wound, a reduction in actin stress fibers parallel to the wound, and a reduction in retrograde actin flow 93 . Triple IF knockdown in astrocytes additionally alters the morphology of adherens junctions (AJs) and decreases the retrograde flow of AJs measured by live imaging of N-cadherin and loss of vinculin localization to AJs 93 . Finally, the astrocyte IF system restricted the mechanical coupling of focal adhesion to the actomyosin network. Given the interdependent nature of astrocyte IFs and the triple IF knockdown strategy used in this study, it is not possible to assign a specific role of vimentin per se. However, in light of the additional studies supporting similar roles of vimentin in other cell types, vimentin is a likely key regulator of astrocyte migration. Overall, these findings may have functional implications for gliomas, since high vimentin expression is an independent prognostic factor for their metastatic aggressiveness 95 .
Vimentin promotes cell migration by enhancing contactdependent cell stiffening Upregulation of vimentin in epithelial cells, in addition to increasing cell motility 96 , induces physical changes in cell shape, loss of cell-cell contacts, and increased turnover of focal adhesions 48 . Furthermore, vimentin supports cellular elasticity and protects against mechanical stress, such as compression 97 .
Tumor cells experience significant compressive stress as they grow, which is known to promote cell migration and invasion related to the formation of new leader cells and actomyosinindependent cell extensions in breast cancer cells 98 .
Using a number of biophysical methods coupled with cell migration assays under low-and high-cell-density conditions, Messica et al. showed that vimentin controls cell migration in dense, but not sparse, cultures 99 . Using the invasive breast carcinoma MDA-MB-231 cells as a model system, the authors compared how the presence or absence of vimentin regulates their mechanical, migratory, and invasive properties. Vimentinlacking MDA-MB-231 cells were softer and more deformable 99 , which are characteristics attributed to more invasive and metastatic cancer cells 100 . Interestingly, the loss of vimentin significantly diminished the ability of MDA-MB-231 cells to migrate and invade in dense, but not sparse, cultures, while vimentin expression positively correlated with longer persistence time of migration 99 . The latter is in line with the previous study supporting a role for vimentin in microtubule-dependent cell polarity regulation during migration 66 . The authors proposed that the decreased migration and invasiveness of the "softer" vimentin-negative MDA-MB-231 cells relate to their deformability in crowded spaces, such that each cell can be molded to accommodate neighboring cells, losing its polarity in the course of this process. In the presence of vimentin, the cells are able to stiffen and redirect their migration to move toward vacant intercellular spaces. It would be intriguing to explore whether and how vimentin regulates cytoskeleton reorganization and cellular stiffening during cancer cell migration through soft substrates, as was recently reported 101 .

Conclusions
Vimentin is a key component of the cytoskeleton with important biological functions at the cellular and organismal levels.
Vimentin is particularly important during development and in cancer during EMT and metastasis. Vimentin interacts with, and regulates, microtubules, actin, focal adhesions, and AJs during cell migration. Recent studies highlight that environmental factors, such as cell density and substrate stiffness, should be carefully considered when studying the role of vimentin in cell migration in vitro. Overexpression and tagging of vimentin can cause defects in the filament network, so novel gene editing strategies at endogenous loci should be used to determine the importance of specific vimentin residues and their respective modifications in filament dynamics. One such approach could be to focus on frequently reported PTM sites on vimentin that have been curated by the comprehensive PhosphoSitePlus database 116 but not validated via mechanistic studies. This approach was applied previously on keratin 8 to reveal conserved tyrosine phosphorylation as an important regulator of solubility and filament dynamics 117 . Future development of small molecules that selectively target vimentin and control the assembly state of vimentin filaments will be essential to understand vimentin dynamics and to target its function as a means to modulate cell migration.
reticulum cells of human lymph nodes, tonsils, and spleen. Differentiation.