Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin.

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called 'ACTIF' that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.

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.

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.

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.

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.

Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures.

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.

Deciphering the transport of elastic filaments by antagonistic motor proteins.

Intermediate filaments are long elastic fibers that are transported by the microtubule-associated motor proteins kinesin and dynein inside the cell. How elastic filaments are efficiently transported by antagonistic motors is not well understood and is difficult to measure with current experimental techniques. Adapting the tug-of-war paradigm for vesiclelike cargos, we develop a mathematical model to describe the motion of an elastic filament punctually bound to antagonistic motors. As observed in cells, up to three modes of transport are obtained; dynein-driven retrograde, kinesin-driven anterograde fast motions, and a slow motion. Motor properties and initial conditions that depend on intracellular context regulate the transport of filaments. Filament elasticity is found to affect both the mode and the efficiency of transport. We further show that the coordination of motors along the filament emerges from the interplay between intracellular context and elastic properties of filaments.

Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and astrocyte migration.

Microtubules play a crucial role in mesenchymal migration by controlling cell polarity and the turnover of cell adhesive structures on the extracellular matrix. The polarized functions of microtubules imply that microtubules are locally regulated. Here, we investigated the regulation and role of two major tubulin post-translational modifications, acetylation and detyrosination, which have been associated with stable microtubules. Using primary astrocytes in a wound healing assay, we show that these tubulin modifications are independently regulated during cell polarization and differently affect cell migration. In contrast to microtubule detyrosination, αTAT1 (ATAT1)-mediated microtubule acetylation increases in the vicinity of focal adhesions and promotes cell migration. We further demonstrate that αTAT1 increases focal adhesion turnover by promoting Rab6-positive vesicle fusion at focal adhesions. Our results highlight the specificity of microtubule post-translational modifications and bring new insight into the regulatory functions of tubulin acetylation.This article has an associated First Person interview with the first author of the paper.

Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport.

Intermediate filaments are a key component of the cytoskeleton. Their transport along microtubules plays an essential role in the control of the shape and structural organization of cells. To identify the key parameters responsible for the control of intermediate filament transport, we generated a model of elastic filament transport by microtubule-associated dynein and kinesin. The model is also applicable to the transport of any elastically-coupled cargoes. We investigate the effect of filament properties such as number of motor binding sites, length, and elasticity on motion of filaments. Additionally, we consider the effect of motor properties, i.e. off rates, on filament transport. When one motor has a catch bond off rate it dictates the motion, whereas when motors have the same type of off rate filaments can alternate between retrograde and anterograde motions. The elasticity of filaments optimizes the filament transport and the coordination of motors along the length of the filament.

Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts.

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell-cell interactions.

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy.

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

Regulation of microtubule-associated motors drives intermediate filament network polarization.

Intermediate filaments (IFs) are key players in the control of cell morphology and structure as well as in active processes such as cell polarization, migration, and mechanoresponses. However, the regulatory mechanisms controlling IF dynamics and organization in motile cells are still poorly understood. In this study, we investigate the mechanisms leading to the polarized rearrangement of the IF network along the polarity axis. Using photobleaching and photoconversion experiments in glial cells expressing vimentin, glial fibrillary acidic protein, and nestin, we show that the distribution of cytoplasmic IFs results from a continuous turnover based on the cooperation of an actin-dependent retrograde flow and anterograde and retrograde microtubule-dependent transports. During wound-induced astrocyte polarization, IF transport becomes directionally biased from the cell center toward the cell front. Such asymmetry in the transport is mainly caused by a Cdc42- and atypical PKC-dependent inhibition of dynein-dependent retrograde transport. Our results show how polarity signaling can affect the dynamic turnover of the IF network to promote the polarization of the network itself.

Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones.

Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.

Single-molecule imaging in live cell using gold nanoparticles.

Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.

Intermediate filaments in cell migration and invasion: the unusual suspects.

Cell migration is a multistep process which relies on the coordination of cytoskeletal structures in space and time. While the roles of actin and microtubules have been investigated in great details, the lack of inhibitors and visualizing tools and the large number of proteins forming intermediate filaments (IFs) have delayed the characterization of IF functions during migration. However, a large body of evidence has progressively pointed to changes in IF composition as an important parameter in the regulation of cell migratory properties both during development and tumor invasion. More recent in-depth analyses show that IFs are dynamically reorganized to participate, together with microfilaments and microtubules, to the key steps leading to cell migration.

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.

Advances in live-cell single-particle tracking and dynamic super-resolution imaging.

Resolving the movement of individual molecules in living cells by single particle tracking methods has allowed many molecular behaviors to be deciphered over the past three decades. These methods have increasingly benefited from advances in microscopy of single nano-objects such as fluorescent dye molecules, proteins or nanoparticles as well as tiny absorbing metal nanoparticles. In parallel to these efforts aiming at tracking ever smaller and more photostable nano-objects in living cells, the development of localization-based super-resolution imaging provided means to increase the number of single molecules tracked on a single cell. In this review we will present the most recent advances in the field.

A highly specific gold nanoprobe for live-cell single-molecule imaging.

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used green fluorescent proteins (GFPs) with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.