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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., (© 2022 Deb Roy et al.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell adhesion to the extracellular matrix is essential for cellular processes, such as migration and invasion. In response to cues from the microenvironment, integrin-mediated adhesions alter cellular behaviour through cytoskeletal rearrangements. The tight association of the actin cytoskeleton with adhesive structures has been extensively studied, whereas the microtubule network in this context has gathered far less attention. In recent years, however, microtubules have emerged as key regulators of cell adhesion and migration through their participation in adhesion turnover and cellular signalling. In this Review, we focus on the interactions between microtubules and integrin-mediated adhesions, in particular, focal adhesions and podosomes. Starting with the association of microtubules with these adhesive structures, we describe the classical role of microtubules in vesicular trafficking, which is involved in the turnover of cell adhesions, before discussing how microtubules can also influence the actin-focal adhesion interplay through RhoGTPase signalling, thereby orchestrating a very crucial crosstalk between the cytoskeletal networks and adhesions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Intermediate filaments (IFs) are one of the three major elements of the cytoskeleton. Their stability, intrinsic mechanical properties, and cell type-specific expression patterns distinguish them from actin and microtubules. By providing mechanical support, IFs protect cells from external forces and participate in cell adhesion and tissue integrity. IFs form an extensive and elaborate network that connects the cell cortex to intracellular organelles. They act as a molecular scaffold that controls intracellular organization. However, IFs have been revealed as much more than just rigid structures. Their dynamics is regulated by multiple signaling cascades and appears to contribute to signaling events in response to cell stress and to dynamic cellular functions such as mitosis, apoptosis, and migration.

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.

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., (© 2017 Leduc and Etienne-Manneville.)

Centrosome positioning is crucial during cell division, cell differentiation, and for a wide range of cell-polarized functions including migration. In multicellular organisms, centrosome movement across the cytoplasm is thought to result from a balance of forces exerted by the microtubule-associated motor dynein. However, the mechanisms regulating dynein-mediated forces are still unknown. We show here that during wound-induced cell migration, the small G protein Cdc42 acts through the polarity protein Dlg1 to regulate the interaction of dynein with microtubules of the cell front. Dlg1 interacts with dynein via the scaffolding protein GKAP and together, Dlg1, GKAP, and dynein control microtubule dynamics and organization near the cell cortex and promote centrosome positioning. Our results suggest that, by modulating dynein interaction with leading edge microtubules, the evolutionary conserved proteins Dlg1 and GKAP control the forces operating on microtubules and play a fundamental role in centrosome positioning and cell polarity.

T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38.

Microtubules are highly dynamic structures whose regulation is crucial for cell division, cell polarity, cell migration, or neuronal differentiation. Because they contribute to most cellular functions, they must be regulated in response to extracellular and intracellular signals. The parameters of microtubule dynamics are numerous and complex and the connection between signaling pathways and regulation of microtubule dynamics remain obscure. Recent observations reveal key players that can both integrate the diversity of signaling cascades and directly influence microtubule dynamics. I review here how modifications of the tubulin dimer, tubulin modifying enzymes, and microtubule-associated proteins are directly involved in the regulation of microtubule behavior and functions., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Centrosome positioning is tightly controlled throughout the cell cycle and probably shares common regulatory mechanisms with spindle-pole positioning. In this article, we detail the possible mechanisms controlling centrosome and spindle positioning in various organisms both in interphase and mitotic cells, and discuss recent findings showing how microtubule plus-end-associated proteins interact with the cell cortex. We suggest that microtubule plus-end complexes simultaneously regulate microtubule dynamics and microtubule anchoring at the cell periphery to allow proper centrosome and spindle-pole positioning.

The cytoskeleton is composed of three distinct elements: actin microfilaments, microtubules and intermediate filaments. The actin cytoskeleton is thought to provide protrusive and contractile forces, and microtubules to form a polarized network allowing organelle and protein movement throughout the cell. Intermediate filaments are generally considered the most rigid component, responsible for the maintenance of the overall cell shape. Cytoskeletal elements must be coordinately regulated for the cell to fulfill complex cellular functions, as diverse as cell migration, cell adhesion and cell division. Coordination between cytoskeletal elements is achieved by signaling pathways, involving common regulators such as the Rho guanosine-5'-triphosphatases (GTPases). Furthermore, evidence is now accumulating that cytoskeletal elements participate in regulating each other. As a consequence, although their functions seem well defined, they are in fact overlapping, with actin playing a role in membrane trafficking and microtubules being involved in the control of protrusive and contractile forces. This cytoskeletal crosstalk is both direct and mediated by signaling molecules. Cell motility is a well-studied example where the interplay between actin and microtubules appears bidirectional. This leads us to wonder which, if any, cytoskeletal element leads the way.

The role of cytoskeletal elements in regulating transport and docking steps that precede exocytosis of secretory organelles is not well understood. We have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the three-dimensional motions of secretory organelles near the plasma membrane in living endothelial cells. Weibel-Palade bodies (WPb), the large tubular storage organelles for von Willebrand factor, were labelled with Rab27a-GFP. By contrast, green fluorescent protein (GFP)-tagged tissue-type plasminogen activator (tPA-GFP) labelled submicron vesicular organelles. Both populations of GFP-labelled organelles underwent stimulated exocytosis. The movement of these morphologically distinct organelles was measured within the evanescent field that penetrated the first 200 nm above the plasma membrane. WPb and tPA-GFP vesicles displayed long-range bidirectional motions and short-range diffusive-like motions. Rotating and oscillating WPb were also observed. TIRF microscopy enabled us to quantify the contribution of actin and microtubules and their associated motors to the organelle motions close to the plasma membrane. Long-range motions, as well as WPb rotations and oscillations, were microtubule-and kinesin-dependent. Disruption of the actin cytoskeleton and inhibition of myosin motors increased the number of long-range motions and, in the case of WPb, their velocity. The actin and microtubules had opposite effects on the mobility of organelles undergoing short-range motions. Actin reduced the mobility and range of motion of both WPb and tPA vesicles, whereas microtubules and kinesin motors increased the mobility of WPb. The results show that the dynamics of endothelial secretory organelles close to the plasma membrane are controlled by the opposing roles of the microtubule and actin cytoskeletal transport systems.