The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional γ-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon”. Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

The polarity protein Pard3 is required for centrosome positioning during neurulation

Microtubules are essential regulators of cell polarity, architecture and motility. The organization of the microtubule network is context-specific. In non-polarized cells, microtubules are anchored to the centrosome and form radial arrays. In most epithelial cells, microtubules are noncentrosomal, align along the apico-basal axis and the centrosome templates a cilium. It follows that cells undergoing mesenchyme-to-epithelium transitions must reorganize their microtubule network extensively, yet little is understood about how this process is orchestrated. In particular, the pathways regulating the apical positioning of the centrosome are unknown, a central question given the role of cilia in fluid propulsion, sensation and signaling. In zebrafish, neural progenitors undergo progressive epithelialization during neurulation, and thus provide a convenient in vivo cellular context in which to address this question. We demonstrate here that the microtubule cytoskeleton gradually transitions from a radial to linear organization during neurulation and that microtubules function in conjunction with the polarity protein Pard3 to mediate centrosome positioning. Pard3 depletion results in hydrocephalus, a defect often associated with abnormal cerebrospinal fluid flow that has been linked to cilia defects. These findings thus bring to focus cellular events occurring during neurulation and reveal novel molecular mechanisms implicated in centrosome positioning.     

An insider's guide to the microtubule cytoskeleton of Giardia

Giardia intestinalis is a zoonotic, parasitic protist with a complex microtubule cytoskeleton critical for motility, attachment, intracellular transport, cell division and transitioning between its two life cycle stages – the cyst and the trophozoite. This review focuses on the structures of the primary elements of the microtubule cytoskeleton and cytoskeletal dynamics throughout this complex giardial life cycle. The giardial cytoskeleton has both highly dynamic elements and more stable MT structures, including several novel structures like the ventral disc that change conformation via unknown mechanisms. While our knowledge of the giardial cytoskeleton is primarily cytological, the completed Giardia genome and recently developed reverse genetic tools affords an opportunity to uncover the mechanisms of Giardia's cytoskeletal dynamics. Fundamental areas of giardial cytoskeletal biology remain to be explored, including high resolution imaging and compositional characterization of cytoskeletal structures required for elucidating the molecular mechanisms of cytoskeletal functioning.     

The conserved mobility of mitochondria during leaf senescence reflects differential regulation of the cytoskeletal components in Arabidopsis thaliana

Leaf senescence is an organized process, which requires fine tuning between nuclear gene expression, activity of proteases and the maintenance of primary metabolism. Recently, we reported that leaf senescence was accompanied by an early degradation of the microtubule cytoskeleton in Arabidopsis thaliana. As the cytoskeleton is essential for cell stability, vesicle shuttling and organelle mobility, it might be asked how the regulation of these cell functions occurs with such drastic modifications of the cytoskeleton. Based on confocal laser microscopy observations and a micro-array analysis, the following addendum shows that mitochondrial mobility is conserved until the late stages of leaf senescence and provides evidences that the actin-cytoskeleton is maintained longer than the microtubule network. This conservation of actin-filaments is discussed with regards to energy metabolism as well as calcium signaling during programmed cell death.Key words: actin, cytoskeleton, microtubule, mitochondria, mobility, senescence     

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.     

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.     

Cytoskeleton organisation during the infection of three brown algal species,Ectocarpus siliculosus,Ectocarpus crouaniorum and Pylaiella littoralis,by the intracellular marine oomycete Eurychasma dicksonii

Oomycete diseases in seaweeds are probably widespread and of significant ecological and economic impact, but overall still poorly understood. This study investigates the organisation of the cytoskeleton during infection of three brown algal species, Pylaiella littoralis, Ectocarpus siliculosus, and Ectocarpus crouaniorum, by the basal marine oomycete Eurychasma dicksonii. Immunofluorescence staining of tubulin revealed how the development of this intracellular biotrophic pathogen impacts on microtubule (MT) organisation of its algal host. The host MT cytoskeleton remains normal and organised by the centrosome until very late stages of the infection. Additionally, the organisation of the parasite's cytoskeleton was examined. During mitosis of the E. dicksonii nucleus the MT focal point (microtubule organisation centre, MTOC, putative centrosome) duplicates and each daughter MTOC migrates to opposite poles of the nucleus. This similarity in MT organisation between the host and pathogen reflects the relatively close phylogenetic relationship between oomycetes and brown algae. Moreover, actin labelling with rhodamine‐phalloidin in E. dicksonii revealed typical images of actin dots connected by fine actin filament bundles in the cortical cytoplasm. The functional and phylogenetic implications of our observations are discussed.     

Cytoskeletal cross-talk in the control of T cell antigen receptor signaling

T cell antigen receptor signaling is triggered and controlled in specialized cellular interfaces formed between T cells and antigen-presenting cells named immunological synapses. Both microtubules and actin cytoskeleton rearrange at the immunological synapse in response to T cell receptor triggering, ensuring in turn the accuracy of intracellular signaling. Recent reports show that the cross-talk between the cortical actin cytoskeleton and microtubule networks is key for structuring the immunological synapse and for controlling T cell receptor signaling. Immunological synapse architecture and the interaction between the signaling machinery and various cytoskeletal elements are therefore crucial for the fine-tuning of T cell signaling.     

Actin-microtubule interactions in the algaNitella: analysis of the mechanism by which microtubule depolymerization potentiates cytochalasin's effects on streaming

Summary In the characean algaNitella, depolymerization of microtubules potentiates the inhibitory effects of cytochalasins on cytoplasmic streaming. Microtubule depolymerization lowers the cytochalasin B and D concentrations required to inhibit streaming, accelerates inhibition and delays streaming recovery. Because microtubule depolymerization does not significantly alter³H-cytochalasin B uptake and release, elevated intracellular cytochalasin concentrations are not the basis for potentiation. Instead, microtubule depolymerization causes actin to become more sensitive to cytochalasin. This increased sensitivity of actin is unlikely to be due to direct stabilization of actin by microtubules, however, because very few microtubules colocalize with the subcortical actin bundles that generate streaming. Furthermore, microtubule reassembly, but not recovery of former transverse alignment, is sufficient for restoring the normal cellular responses to cytochalasin D. We hypothesize that either tubulin or microtubule-associated proteins, released when microtubules depolymerize, interact with the actin cytoskeleton and sensitize it to cytochalasin.Abbreviations APW artificial pond water - Ca_c cytoplasraic free calcium concentration - DMSO dimethyl sulfoxide - MT microtubule-minus - MT+ microtubule-plus.     

MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons.     

Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation.     

Purposive behavior and cognitive mapping: a neural network model

This study presents a real-time, biologically plausible neural network approach to purposive behavior and cognitive mapping. The system is composed of (a) an action system, consisting of a goal-seeking neural mechanism controlled by a motivational system; and (b) a cognitive system, involving a neural cognitive map. The goal-seeking mechanism displays exploratory behavior until either (a) the goal is found or (b) an adequate prediction of the goal is generated. The cognitive map built by the network is a top logical map, i.e., it represents only the adjacency, but not distances or directions, between places. The network has recurrent and non-recurrent properties that allow the reading of the cognitive map without modifying it. Two types of predictions are introduced: fast-time and real-time predictions. Fast-time predictions are produced in advance of what occurs in real time, when the information stored in the cognitive map is used to predict the remote future. Real-time predictions are generated simultaneously with the occurrence of environmental events, when the information stored in the cognitive map is being updated. Computer simulations show that the network successfully describes latent learning and detour behavior in rats. In addition, simulations demonstrate that the network can be applied to problem-solving paradigms such as the Tower of Hanoi puzzle.     

Lipopolysaccharide induces distinct alterations in the microtubule cytoskeleton of monocytes

Microtubules are obligate functional elements of almost all eukaryotic cells. They are involved in a broad range of essential cellular functions and structural changes of this system may trigger cell death. Recently, we have reported that lipopolysaccharides inhibitin vitro microtubule formation due to exclusion of microtubule-associated proteins. The distinct epitopes of lipopolysaccharides responsible for these effects and thein vivo relevance of these data are unknown. Therefore, this study was conducted to elucidate the effects of lipid A, the biologically active motif of lipopolysaccharides, on microtubule formationin vitro and to prove whether lipopolysaccharides affect the microtubule architecture of cultured human monocytesin vivo. Despite a dose- and pH-dependent inhibition of microtubule formation by lipopolysaccharides, inhibition of microtubule assembly could be mimicked by lipid A. Near-infrared two-photon microscopy revealed that human peripheral blood monocytes accumulate lipopolysaccharides. A vesicular distribution pattern of lipopolysaccharides within the monocytes was observed. Confocal laser scanning microscopy demonstrated alterations in the microtubule architecture of monocytes after incubation with lipopolysaccharides. Lipid A seems to be responsible for the observed crosstalk between lipopolysaccharides and microtubule proteins. Furthermore, our data indicate that lipopolysaccharides may affect the microtubule architecture in human monocytes after intracellular accumulation directly. Therefore, we conclude, that the microtubule cytoskeleton is an essential intracellular target for sepsis-relevant bacterial components such as lipopolysaccharides. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mechanical model of cytoskeleton structuration during cell adhesion and spreading

The biomechanical behavior of an adherent cell is intimately dependent on its cytoskeleton structure. Several models have been proposed to study this structure taking into account its existing internal forces. However, the structural and geometrical complexities of the cytoskeleton's filamentous networks lead to difficulties for determining a biologically realistic architecture. The objective of this paper is to present a mechanical model, combined with a numerical method, devoted to the form-finding of the cytoskeleton structure (shape and internal forces) when a cell adheres on a substrate. The cell is modeled as a granular medium, using rigid spheres (grains) corresponding to intracellular cross-linking proteins and distant mechanical interactions to reproduce the cytoskeleton filament internal forces. At the initial state (i.e., before adhesion), these interactions are tacit. The adhesion phenomenon is then simulated by considering microtubules growing from the centrosome towards transmembrane integrin-like receptors. The simulated cell shape changes in this process and results in a mechanically equilibrated structure with traction and compression forces, in interaction with the substrate reactions. This leads to a compressive microtubule network and a corresponding tensile actin-filament network. The results provide coherent shape and forces information for developing a mechanical model of the cytoskeleton structure, which can be exploitable in future biomechanical studies of adherent cells.     

Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

Background

TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.

Methodology and Principal Findings

We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca²⁺-influx via TRPV4.

Conclusions and Significance

TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.     

T lymphocytes and neutrophil granulocytes differ in regulatory signaling and migratory dynamics with regard to spontaneous locomotion and chemotaxis

Chemotactic migration of T lymphocytes and neutrophil granulocytes within a three-dimensional collagen matrix is distinct from spontaneous, matrix-induced migration concerning dynamic parameters and regulatory intracellular signaling. Both spontaneous T lymphocyte locomotion and stromal-cell-derived factor-1 (SDF-1)-induced chemotaxis-involved protein tyrosine kinase (PTK) activity, whereas only SDF-1-induced migration was protein kinase C (PKC) dependent. Spontaneous locomotion of neutrophil granulocytes was independent of PKC and PTK activity, but formyl-methionyl-leucyl-phenylalanine-induced migration involved PKC activity. In addition, the microtubule cytoskeleton was not changed after induction of chemotaxis in both cell types. T lymphocytes had a well-developed microtubule cytoskeleton with the microtubule organizing center located in the uropod, whereas neutrophil granulocytes revealed a clustered tubulin distribution at the leading edge of the migrating cell. Therefore, differences of the microtubule cytoskeleton might contribute to differences in locomotion between T lymphocytes and neutrophil granulocytes but not to differences between spontaneous locomotion and chemotaxis.     

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini‐organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high‐pressure frozen and freeze‐substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule‐dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule‐independent strain, progeny virions lacked organisation. Conversely to the microtubule‐dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome‐less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule‐dependent strain resulted in a significant increase of genome‐less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.     

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.     

Molecular aspects of microtubule dynamics in plants

Microtubules are highly dynamic structures that play a major role in a wide range of processes, including cell morphogenesis, cell division, intracellular transport and signaling. The recent identification in plants of proteins involved in microtubule organization has begun to reveal how cytoskeleton dynamics are controlled.