Dystonin/BPAG1 Promotes Plus-End-Directed Transport of Herpes Simplex Virus 1 Capsids on Microtubules during Entry

During infection by herpes simplex virus 1 (HSV-1), the viral capsid is transported around the cytoplasm along the microtubule (MT) network. Although molecular motors have been implicated in this process, the composition of the molecular machinery required for efficient directional transport is unknown. We previously showed that dystonin (BPAG1) is recruited to HSV-1 capsids by the capsid-bound tegument protein pUL37 to promote efficient cytoplasmic transport of capsids during egress. Dystonin is a cytoskeleton cross-linker which localizes at MT plus ends and has roles in retrograde and anterograde transport in neurons. In this study, we investigated the role of dystonin during the entry stages of HSV-1 infection. Because of the way in which the MT network is organized, capsids are required to change their direction of motion along the MTs as they travel from the point of entry to the nucleus, where replication takes place. Thus, capsids first travel to the centrosome (the principal microtubule organizing center) by minus-end-directed transport and then switch polarity and travel to the nucleus by plus-end-directed transport. We observed that transport of capsids toward the centrosome was slowed, but not blocked, by dystonin depletion. However, transport of capsids away from the centrosome was significantly impaired, causing them to accumulate in the vicinity of the centrosome and reducing the numbers reaching the nucleus. We conclude that, during entry of HSV-1, dystonin has a specific role in plus-ended transport of capsids from the centrosome to the nucleus.     

Herpesvirus Tegument Protein pUL37 Interacts with Dystonin/BPAG1 To Promote Capsid Transport on Microtubules during Egress

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that travels long distances through cells using the microtubule network. Its 125-nm-diameter capsid is a large cargo which efficiently recruits molecular motors for movement. Upon entry, capsids reach the centrosome by minus-end-directed transport. From there, they are believed to reach the nucleus by plus-end-directed transport. Plus-end-directed transport is also important during egress, when capsids leave the nucleus to reach the site of envelopment in the cytoplasm. Although capsid interactions with dynein and kinesins have been described in vitro, the actual composition of the cellular machinery recruited by herpesviruses for capsid transport in infected cells remains unknown. Here, we identify the spectraplakin protein, dystonin/BPAG1, an important cytoskeleton cross-linker involved in microtubule-based transport, as a binding partner of the HSV-1 protein pUL37, which has been implicated in capsid transport. Viral replication is delayed in dystonin-depleted cells, and, using video microscopy of living infected cells, we show that dystonin depletion strongly inhibits capsid movement in the cytoplasm during egress. This study provides new insights into the cellular requirements for HSV-1 capsid transport and identifies dystonin as a nonmotor protein part of the transport machinery.     

Microtubule self-repair

The stochastic switching between microtubule growth and shrinkage is a fascinating and unique process in the regulation of the cytoskeleton. To understand it, almost all attention has been focused on the microtubule ends. However, recent research has revived the idea that tubulin dimers can also be exchanged in protofilaments along the microtubule shaft, thus repairing the microtubule and protecting it from disassembly. Here, we review the research describing this phenomenon, the mechanisms regulating the removal and insertion of tubulin dimers, as well as the potential implications for key functions of the microtubule network, such as intracellular transport and cell polarization.     

How cellular membranes can regulate microtubule network

Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing “tracks” for their subsequent transport.     

Disruption of microtubules in rat skeletal muscle does not inhibit insulin- or contraction-stimulated glucose transport

Insulin and muscle contractions stimulate glucose transport in skeletal muscle through a translocation of intracellular GLUT4 glucose transporters to the cell surface. Judged by immunofluorescence microscopy, part of the GLUT4 storage sites is associated with the extensive microtubule cytoskeleton found in all muscle fibers. Here, we test whether microtubules are required mediators of the effect of insulin and contractions. In three different incubated rat muscles with distinct fiber type composition, depolymerization of microtubules with colchicine for < or =8 h did not inhibit insulin- or contraction-stimulated 2-deoxyglucose transport or force production. On the contrary, colchicine at least partially prevented the approximately 30% decrease in insulin-stimulated transport that specifically developed during 8 h of incubation in soleus muscle but not in flexor digitorum brevis or epitrochlearis muscles. In contrast, nocodazole, another microtubule-disrupting drug, rapidly and dose dependently blocked insulin- and contraction-stimulated glucose transport. A similar discrepancy between colchicine and nocodazole was also found in their ability to block glucose transport in muscle giant "ghost" vesicles. This suggests that the ability of insulin and contractions to stimulate glucose transport in muscle does not require an intact microtubule network and that nocodazole inhibits glucose transport independently of its microtubule-disrupting effect.     

New roles for perforins and proteases in apicomplexan egress

Egress is a pivotal step in the life cycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late-stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called serine-repeat antigens that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.     

Cytoskeletal involvement in neuronal learning: a review

This paper introduces the ideas of neural networks in the context of currently recognized cellular structures within neurons. Neural network models and paradigms require adaptation of synapses for learning to occur in the network. Some models of learning paradigms require information to move from axon to dendrite. This motivated us to examine the possibility of intracellular signaling to mediate such signals. The cytoskeleton forms a substrate for intracellular signaling via material transport and other putative mechanisms. Furthermore, many experimental results suggest a link between the cytoskeleton and cognitive processing. In this paper we review research on intracellular signaling in the context of neural network learning.Abbreviations MT microtubule - MTs microtubules - ART adaptive resonance theory - RCE restricted coulomb energy - MAP microtubule associated protein - NO nitric oxide Correspondence to: J. Dayhoff     

Basis for MAP4 dephosphorylation-related microtubule network densification in pressure overload cardiac hypertrophy

Increased activity of Ser/Thr protein phosphatases types 1 (PP1) and 2A (PP2A) during maladaptive cardiac hypertrophy contributes to cardiac dysfunction and eventual failure, partly through effects on calcium metabolism. A second maladaptive feature of pressure overload cardiac hypertrophy that instead leads to heart failure by interfering with cardiac contraction and intracellular transport is a dense microtubule network stabilized by decoration with microtubule-associated protein 4 (MAP4). In an earlier study we showed that the major determinant of MAP4-microtubule affinity, and thus microtubule network density and stability, is site-specific MAP4 dephosphorylation at Ser-924 and to a lesser extent at Ser-1056; this was found to be prominent in hypertrophied myocardium. Therefore, in seeking the etiology of this MAP4 dephosphorylation, we looked here at PP2A and PP1, as well as the upstream p21-activated kinase 1, in maladaptive pressure overload cardiac hypertrophy. The activity of each was increased persistently during maladaptive hypertrophy, and overexpression of PP2A or PP1 in normal hearts reproduced both the microtubule network phenotype and the dephosphorylation of MAP4 Ser-924 and Ser-1056 seen in hypertrophy. Given the major microtubule-based abnormalities of contractile and transport function in maladaptive hypertrophy, these findings constitute a second important mechanism for phosphatase-dependent pathology in the hypertrophied and failing heart.     

Microtubule organization and function in epithelial cells

Microtubules are essential for many aspects of polarity in multicellular organisms, ranging from the asymmetric distribution of cell-fate determinants in the one-cell embryo to the transient polarity generated in migrating fibroblasts. Epithelial cells exhibit permanent cell polarity characterized by apical and basolateral surface domains of distinct protein and lipid composition that are segregated by tight junctions. They are also endowed with a microtubule network that reflects the asymmetry of their cell surface: microtubule minus-ends face the apical- and microtubule plus-ends the basal domain. Strikingly, the formation of distinct surface domains during epithelial differentiation is accompanied by the re-organization of microtubules from a uniform array focused at the centrosome to the noncentrosomal network that aligns along the apico-basolateral polarity axis. The significance of this coincidence for epithelial morphogenesis and the signaling mechanisms that drive microtubule repolymerization in developing epithelia remain major unresolved questions that we are only beginning to address. Studies in cultured polarized epithelial cells have established that microtubules serve as tracks that facilitate targeted vesicular transport. Novel findings suggest, moreover, that microtubule-based transport promotes protein sorting, and even the generation of transport carriers in the endo- and exocytic pathways.     

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 .     

EB-family proteins: Functions and microtubule interaction mechanisms

Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.     

Ordering microtubules

How do cells order their cytoplasm? While microtubule organizing centers have long been considered essential to conferring order by virtue of their microtubule nucleating activity, attention has currently refocused on the role that microtubule motors play in organizing microtubules. An intriguing set of recent findings⁽¹⁾ reveals that cell fragments, lacking microtubule organizing centers, rapidly organize microtubules into a radial array during organelle transport driven by the microtubule motor, cytoplasmic dynein. Further, interaction of radial microtubules with the cell surface centers the array, revealing that centering information resides not with centrosomes but with organized microtubules.     

Involvement of cytoskeletal components in BK virus infectious entry

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.     

Biogenesis of N-Cadherin-dependent Cell-Cell Contacts in Living Fibroblasts Is a Microtubule-dependent Kinesin-driven Mechanism

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin-dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin-dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.     

Melanosomes on the move: a model to understand organelle dynamics

Advances in live-cell microscopy have revealed the extraordinarily dynamic nature of intracellular organelles. Moreover, movement appears to be critical in establishing and maintaining intracellular organization and organellar and cellular function. Motility is regulated by the activity of organelle-associated motor proteins, kinesins, dyneins and myosins, which move cargo along polar MT (microtubule) and actin tracks. However, in most instances, the motors that move specific organelles remain mysterious. Over recent years, pigment granules, or melanosomes, within pigment cells have provided an excellent model for understanding the molecular mechanisms by which motor proteins associate with and move intracellular organelles. In the present paper, we discuss recent discoveries that shed light on the mechanisms of melanosome transport and highlight future prospects for the use of pigment cells in unravelling general molecular mechanisms of intracellular transport.     

How does Legionella pneumophila exit the host cell?

In recent years, tremendous progress has been made in unraveling the elegant mechanisms by which intracellular pathogens invade host cells and establish intracellular infections. By contrast, our knowledge of the mechanisms of host cell cytolysis and the egress of intracellular pathogens is still in its infancy. Temporal pore-formation-mediated lysis of the host and exit by Legionella pneumophila and Leishmania could provide a new model of egress for other intracellular pathogens, many of which exhibit pore-forming or cytolysin activity     

Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons

During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the 'married' model or as non-enveloped capsids suggested by the 'separate' model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the 'separate model' for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles.     

Spatiotemporal modeling and analysis of transient gene delivery

A quantitative and mechanistic understanding of intracellular transport processes in eukaryotic cells during transient transfection is an important prerequisite for the systematic and specific optimization of transient gene expression procedures for pharmaceutic and industrial protein production. There is evidence that intracellular transport processes during gene delivery and their regulation may have significant influence on the transfection efficiency. This contribution describes a compartmented, spatiotemporally resolved and stochastic modeling approach that describes intracellular transport processes responsible for gene delivery during transient transfection. It enables a detailed prediction and analysis and identification of potential bottlenecks. This model is currently being adapted to a model cell line, HEK293s. The simulated results are compared with experimental quantitative polymerase chain reaction (qPCR) data and confocal imaging data obtained with transfected and stained HEK293 cells. Global parameter estimation is performed to qPCR data based on two different novel plasmid constructs in order to identify candidates for plasmid-specific transport parameter variations. The influence of the specific property of HEK293 cells to grow in clusters is investigated and the impact of active microtubule transport depending on cell morphology and clustering is examined. A general sensitivity analysis allows for the identification of the sensitive parameters.     

Electron-microscopic and immunohistochemical study of the reorganization of the microtubule system in granular cells of frog urinary bladder after water transport induction]

Structural changes in organization of the microtubule system in granular cells of frog urinary bladder after water transport induction by vasopressin were studied by methods of electron microscopy and immunocytochemistry. It is shown that in steady-state conditions microtubules form a wide network equally distributed in the whole cytoplasm of granular cells. After vasopressin action, the amount of microtubules increases in the apical region of the cytoplasm. A predominant orientation of microtubules, perpendicular to the apical membrane direction, appears. A structural association of microtubules with specific granules and large vacuoles was observed. A supposition is advanced about association of the described microtubule system reorganization with the activation of vectorial intracellular transport occurring after transepithelial water transport induction.