The distinct localization of CDC42 isoforms is responsible for their specific functions during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), which only differ in their carboxy-terminal sequence, including the CAAX motif essential for their association with membranes. We show that these divergent sequences do not directly affect the range of CDC42's potential binding partners but indirectly influence CDC42-driven signaling by controlling the subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u associates with the leading-edge plasma membrane of migrating cells, where it recruits the Par6-PKCζ complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it regulates N-WASP-mediated endocytosis. Both CDC42 isoforms contribute their specific functions to promote the chemotaxis of neural precursors, demonstrating that their expression pattern is decisive for tissue-specific cell behavior.

Cytoskeletal crosstalk: A focus on intermediate filaments.

The cytoskeleton, comprising actin microfilaments, microtubules, and intermediate filaments, is crucial for cell motility and tissue integrity. While prior studies largely focused on individual cytoskeletal networks, recent research underscores the interconnected nature of these systems in fundamental cellular functions like adhesion, migration, and division. Understanding the coordination of these distinct networks in both time and space is essential. This review synthesizes current findings on the intricate interplay between these networks, emphasizing the pivotal role of intermediate filaments. Notably, these filaments engage in extensive crosstalk with microfilaments and microtubules through direct molecular interactions, cytoskeletal linkers, and molecular motors that form molecular bridges, as well as via more complex regulation of intracellular signaling.

Cell polarity changes in cancer initiation and progression.

Cell polarity, which consists of the morphological, structural, and functional organization of cells along a defined axis, is a feature of healthy cells and tissues. In contrast, abnormal polarity is a hallmark of cancer cells. At the molecular level, key evolutionarily conserved proteins that control polarity establishment and maintenance in various contexts are frequently altered in cancer, but the relevance of these molecular alterations in the oncogenic processes is not always clear. Here, we summarize the recent findings, shedding new light on the involvement of polarity players in cancer development, and discuss the possibility of harnessing cell polarity changes to better predict, diagnose, and cure cancers.

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ-binding motif to the cytoplasm.

The carboxy-terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work, we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by E C-terminal specific antibodies nor by the PDZ domain-containing protein-binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

SCRIB controls apical contractility during epithelial differentiation.

Although mutations in the SCRIB gene lead to multiple morphological organ defects in vertebrates, the molecular pathway linking SCRIB to organ shape anomalies remains elusive. Here, we study the impact of SCRIB-targeted gene mutations during the formation of the gut epithelium in an organ-on-chip model. We show that SCRIB KO gut-like epithelia are flatter with reduced exposed surface area. Cell differentiation on filters further shows that SCRIB plays a critical role in the control of apical cell shape, as well as in the basoapical polarization of myosin light chain localization and activity. Finally, we show that SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 and identify an evolutionary conserved SHROOM binding site in the SCRIB carboxy-terminal that is required for SCRIB function in the control of apical cell shape. Our results demonstrate that SCRIB plays a key role in epithelial morphogenesis by controlling the epithelial apical contractility during cell differentiation.

Models of vimentin organization under actin-driven transport.

Intermediate filaments form an essential structural network, spread throughout the cytoplasm, and play a key role in cell mechanics, intracellular organization, and molecular signaling. The maintenance of the network and its adaptation to the cell's dynamic behavior relies on several mechanisms implicating cytoskeletal crosstalk which are not fully understood. Mathematical modeling allows us to compare several biologically realistic scenarios to help us interpret experimental data. In this study we observe and model the dynamics of the vimentin intermediate filaments in single glial cells seeded on circular micropatterns following microtubule disruption by nocodazole treatment. In these conditions, the vimentin filaments move towards the cell center and accumulate before eventually reaching a steady state. In the absence of microtubule-driven transport, the motion of the vimentin network is primarily driven by actin-related mechanisms. To model these experimental findings, we hypothesize that vimentin may exist in two states, mobile and immobile, and switch between the states at unknown (either constant or nonconstant) rates. Mobile vimentin is assumed to advect with either constant or nonconstant velocity. We introduce several biologically realistic scenarios using this set of assumptions. For each scenario, we use differential evolution to find the best parameter sets resulting in a solution that most closely matches the experimental data and then the assumptions are evaluated using the Akaike information criterion. This modeling approach allows us to conclude that our experimental data are best explained by a spatially dependent trapping of intermediate filaments or a spatially dependent speed of actin-dependent transport.

The diverse actions of cytoskeletal vimentin in bacterial infection and host defense.

Bacterial infection is a major threat to human health, with infections resulting in considerable mortality, urging the need for a more profound understanding of bacteria-host interactions. During infection of cells, host cytoskeletal networks constantly interact with bacteria and are integral to their uptake. Vimentin, an intermediate filament protein, is one such cytoskeletal component that interacts with bacteria during infection. Although vimentin is predominantly present in the cytoplasm, it also appears in a secreted form or at the surface of multiple cell types, including epithelial cells, endothelial cells, macrophages and fibroblasts. As a cytoplasmic protein, vimentin participates in bacterial transportation and the consequential immune-inflammatory responses. When expressed on the cell surface, vimentin can be both pro- and anti-bacterial, favoring bacterial invasion in some contexts, but also limiting bacterial survival in others. Vimentin is also secreted and located extracellularly, where it is primarily involved in bacterial-induced inflammation regulation. Reciprocally, bacteria can also manipulate the fate of vimentin in host cells. Given that vimentin is not only involved in bacterial infection, but also the associated life-threatening inflammation, the use of vimentin-targeted drugs might offer a synergistic advantage. In this Review, we recapitulate the abundant evidence on vimentin and its dynamic changes in bacterial infection and speculate on its potential as an anti-bacterial therapeutic target.

Non-catalytic allostery in α-TAT1 by a phospho-switch drives dynamic microtubule acetylation.

Spatiotemporally dynamic microtubule acetylation underlies diverse physiological and pathological events. Despite its ubiquity, the molecular mechanisms that regulate the sole microtubule acetylating agent, α-tubulin-N-acetyltransferase-1 (α-TAT1), remain obscure. Here, we report that dynamic intracellular localization of α-TAT1 along with its catalytic activity determines efficiency of microtubule acetylation. Specifically, we newly identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements-nuclear export, nuclear import, and cytosolic retention. Their balance is tuned via phosphorylation by CDK1, PKA, and CK2, and dephosphorylation by PP2A. While the unphosphorylated form binds to importins and resides both in cytosol and nucleus, the phosphorylated form binds to specific 14-3-3 adapters and accumulates in the cytosol for maximal substrate access. Unlike other molecules with a similar phospho-regulated signal motif, α-TAT1 uniquely uses the nucleus as a hideout. This allosteric spatial regulation of α-TAT1 function may help uncover a spatiotemporal code of microtubule acetylation in normal and aberrant cell behavior.

Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics.

Fluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes. Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration.

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

PTEN inhibits AMPK to control collective migration.

Pten is one of the most frequently mutated tumour suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterised. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localisation at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness.

Intermediate filaments: Integration of cell mechanical properties during migration.

Cell migration is a vital and dynamic process required for the development of multicellular organisms and for immune system responses, tissue renewal and wound healing in adults. It also contributes to a variety of human diseases such as cancers, autoimmune diseases, chronic inflammation and fibrosis. The cytoskeleton, which includes actin microfilaments, microtubules, and intermediate filaments (IFs), is responsible for the maintenance of animal cell shape and structural integrity. Each cytoskeletal network contributes its unique properties to dynamic cell behaviour, such as cell polarization, membrane protrusion, cell adhesion and contraction. Hence, cell migration requires the dynamic orchestration of all cytoskeleton components. Among these, IFs have emerged as a molecular scaffold with unique mechanical features and a key player in the cell resilience to mechanical stresses during migration through complex 3D environment. Moreover, accumulating evidence illustrates the participation of IFs in signalling cascades and cytoskeletal crosstalk. Teaming up with actin and microtubules, IFs contribute to the active generation of forces required for cell adhesion and mesenchymal migration and invasion. Here we summarize and discuss how IFs integrate mechanical properties and signalling functions to control cell migration in a wide spectrum of physiological and pathological situations.

Impact of noise on the regulation of intracellular transport of intermediate filaments.

Noise affects all biological processes from molecules to cells, organisms and populations. Although the effect of noise on these processes is highly variable, evidence is accumulating which shows natural stochastic fluctuations (noise) can facilitate biological functions. Herein, we investigate the effect of noise on the transport of intermediate filaments in cells by comparing the stochastic and deterministic formalizations of the bidirectional transport of intermediate filaments, long elastic polymers transported along microtubules by antagonistic motor proteins (Dallon et al., 2019; Portet et al., 2019). By numerically exploring discrepancies in timescales and attractors between both formalizations, we characterize the impact of stochastic fluctuations on the individual and ensemble transport. Biologically, we find that noise promotes the collective movement of intermediate filaments and increases the efficiency of its regulation by the biochemical properties of motor-cargo interactions. While stochastic fluctuations reduce the impact of the initial distributions of motor proteins in cells, the number of binding sites and the affinity of motor-cargo interactions are the key parameters controlling transport efficiency and efficacy.

Interactions of Severe Acute Respiratory Syndrome Coronavirus 2 Protein E With Cell Junctions and Polarity PSD-95/Dlg/ZO-1-Containing Proteins.

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ-binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains, which is identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified 10 human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, and LNX2) and MPP5/PALS1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2, and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4, and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in the E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging.

Intermediate filaments (IFs) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49-nanometer axial repeat both in cells and in vitro. As unit-length filaments (ULFs) were measured at ~59 nanometers, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical, and structural properties in cells.

Microtubules tune mechanosensitive cell responses.

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.

Intermediate Filaments from Tissue Integrity to Single Molecule Mechanics.

Cytoplasmic intermediate filaments (IFs), which together with actin and microtubules form the cytoskeleton, are composed of a large and diverse family of proteins. Efforts to elucidate the molecular mechanisms responsible for IF-associated diseases increasingly point towards a major contribution of IFs to the cell's ability to adapt, resist and respond to mechanical challenges. From these observations, which echo the impressive resilience of IFs in vitro, we here discuss the role of IFs as master integrators of cell and tissue mechanics. In this review, we summarize our current understanding of the contribution of IFs to cell and tissue mechanics and explain these results in light of recent in vitro studies that have investigated physical properties of single IFs and IF networks. Finally, we highlight how changes in IF gene expression, network assembly dynamics, and post-translational modifications can tune IF properties to adapt cell and tissue mechanics to changing environments.

Multiscale rheology of glioma cells.

Cells tend to soften during cancer progression, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. Using two microrheology techniques, a single-cell parallel plates rheometer to probe whole-cell mechanics and optical tweezers to probe intracellular rheology, we show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton, and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.

Intermediate filaments.

Cell morphology, architecture and dynamics primarily rely on intracellular cytoskeletal networks, which in metazoans are mainly composed of actin microfilaments, microtubules and intermediate filaments (IFs). The diameter size of 10 nm - intermediate between the diameters of actin microfilaments and microtubules - initially gave IFs their name. However, the structure, dynamics, mechanical properties and functions of IFs are not intermediate but set them apart from actin and microtubules. Because of their nucleotide-independent assembly, the lack of intrinsic polarity, their relative stability and their complex composition, IFs had long been overlooked by cell biologists. Now, the numerous human diseases identified to be associated with IF gene mutations and the accumulating evidence of IF functions in cell and tissue integrity explain the growing attention that is being given to the structural characteristics, dynamics and functions of these filaments. In this Primer, we highlight the growing evidence that has revealed a role for IFs as a key element of the cytoskeleton, providing versatile, tunable, cell-type-specific filamentous networks with unique cytoplasmic and nuclear functions.

Cytoskeletal Crosstalk in Cell Migration.

Cell migration is a highly dynamic process driven by the cytoskeleton, which mainly comprises the actin microfilaments, microtubules, and intermediate filaments. During migration, cells polarize and form protrusions at the front, where new adhesions are formed. These nascent adhesions mature into focal adhesions that transmit the traction forces required for movement. All of these steps are coupled to major cytoskeletal rearrangements and are controlled by a wide array of signaling cascades. The constant crosstalk between actin, microtubules, and intermediate filaments ensures their coordinated dynamics to facilitate cell migration. Here, we first describe how master regulators, such as RhoGTPases, can simultaneously control the three cytoskeletal structures. We then summarize the recent crosstalk mechanisms by which cytoskeletal networks can locally regulate one another in order to function in a coordinated and efficient manner during migration.