Closing the GAP between polarity and vesicle transport

How are tight junctions maintained? In this issue of Cell, Wells et al. (2006) provide intriguing evidence for a new pathway that links polarity proteins and vesicle transport to the maintenance of tight junctions, through the control of Cdc42 by Rich1, a GTPase-activating protein.     

The transport properties of axonal microtubules establish their polarity orientation

It is well established that axonal microtubules (MTs) are uniformly oriented with their plus ends distal to the neuronal cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-665). However, the mechanisms by which these MTs achieve their uniform polarity orientation are unknown. Current models for axon growth differ with regard to the contributions of MT assembly and transport to the organization and elaboration of the axonal MT array. Do the transport properties or assembly properties of axonal MTs determine their polarity orientation? To distinguish between these possibilities, we wished to study the initiation and outgrowth of axons under conditions that would arrest MT assembly while maintaining substantial levels of preexisting polymer in the cell body that could still be transported into the axon. We found that we could accomplish this by culturing rat sympathetic neurons in the presence of nanomolar levels of vinblastine. In concentrations of the drug up to and including 100 nM, the neurons actively extend axons. The vinblastine- axons are shorter than control axons, but clearly contain MTs. To quantify the effects of the drug on MT mass, we compared the levels of polymer throughout the cell bodies and axons of neurons cultured overnight in the presence of 0, 16, and 50 nM vinblastine with the levels of MT polymer in freshly plated neurons before axon outgrowth. Without drug, the total levels of polymer increase by roughly twofold. At 16 nM vinblastine, the levels of polymer are roughly equal to the levels in freshly plated neurons, while at 50 nM, the levels of polymer are reduced by about half this amount. Thus, 16 nM vinblastine acts as a "kinetic stabilizer" of MTs, while 50 nM results in some net MT disassembly. At both drug concentrations, there is a progressive increase in the levels of MT polymer in the axons as they grow, and a corresponding depletion of polymer from the cell body. These results indicate that highly efficient mechanisms exist in the neuron to transport preassembled MTs from the cell body into the axon. These mechanisms are active even at the expense of the cell body, and even under conditions that promote some MT disassembly in the neuron. MT polarity analyses indicate that the MTs within the vinblastine-axons, like those in control axons, are uniformly plus-end-distal.(ABSTRACT TRUNCATED AT 400 WORDS)     

CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle-associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left-right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.     

Evidence that microtubules do not mediate opsin vesicle transport in photoreceptors

The organization of the rod photoreceptor cytoskeleton suggests that microtubules (MTs) and F actin are important in outer segment (OS) membrane renewal. We studied the role of the cytoskeleton in this process by first quantifying OS membrane assembly in rods from explanted Xenopus eyecups with a video assay for disc morphogenesis and then determining if the rate of assembly was reduced after drug disassembly of either MTs or F actin. Membrane assembly was quantified by continuously labeling newly forming rod OS membranes with Lucifer Yellow VS (LY) and following the tagged membranes' distal displacement along the OS. LY band displacement displayed a linear increase over 16 h in culture. These cells possessed a longitudinally oriented network of ellipsoid MTs between the sites of OS protein synthesis and OS membrane assembly. Incubation of eyecups in nocodazole, colchicine, vinblastine, or podophyllotoxin disassembled the ellipsoid MTs. Despite their absence, photoreceptors maintained a normal rate of OS assembly. In contrast, photoreceptors displayed a reduced distal displacement of LY-labeled membranes in eyecups treated with cytochalasin D, showing that our technique can detect drug-induced changes in basal rod outer segment assembly. The reduction noted in the cytochalasin-treated cells was due to the abnormal lateral displacement of newly added OS disc membranes that occurs with this drug (Williams, D. S., K. A. Linberg, D. K. Vaughan, R. N. Fariss, and S. K. Fisher. 1988. J. Comp. Neurol. 272:161-176). Together, our results indicate that the vectorial transport of OS membrane constituents through the ellipsoid and their assembly into OS disc membranes are not dependent on elliposid MT integrity.     

Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm

To assay the detailed structural relationship between axonally transported vesicles and their substrate microtubules, vesicle transport was focally cold blocked in axoplasm that was extruded from the squid giant axon. A brief localized cold block concentrated anterogradely and retrogradely transported vesicles selectively on either the proximal or or distal side of the block. Normal movement of the concentrated vesicles was reactivated by rewarming the cold-blocked axoplasm. At the periphery of the axoplasm, moving vesicles were located on individual microtubules that had become separated from the other cytomatrix components. The presence of moving vesicles on isolated microtubules permitted the identification of the structural components required for vesicle transport along microtubules. The results show that 16-18-nm cross-bridges connect both anterogradely and retrogradely moving vesicles to their substrate microtubules. These observations demonstrate that cross-bridges are fundamental are fundamental components of vesicle transport along axonal microtubules. Thus, vesicle transport can now be included among those cell motile systems such as muscle and axonemes that are based on a cross-bridge-mediated mechanism.     

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.     

Connecting vesicle transport to the cytoskeleton

The cytoskeleton is crucial for the efficient and polarized transport of vesicles in intracellular membrane-sorting pathways. Recent studies have identified specific kinesin, dynein, and myosin motor proteins that mediate defined membrane transport steps. Important clues have also been uncovered about the nature of motor-protein receptors on vesicular cargoes and the molecular mechanisms of motor-protein regulation.     

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.     

Dynamic instability of native microtubules from squid axons is rare and independent of gliding and vesicle transport

Dynamic instability characterizes the steady-state behavior of microtubules in vitro whereby polymer mass remains constant, while individual microtubules in the population may either grow or shrink. Video-enhanced contrast light microscopy was used to directly observe dynamic length changes in native, MAP-containing microtubules from squid axoplasm. We wanted to determine whether dynamic instability characterizes the steady-state behavior of axoplasmic microtubules in vitro. The lengths of a representative population of over 400 microtubules were analyzed. "Dynamic" microtubules were found to represent about 2% of the population. This observation is different from that described for cultured cells or microtubules assembled from PC-purified tubulin where most microtubules were either growing or shrinking.     

Transport of dendritic microtubules establishes their nonuniform polarity orientation

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus- ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.     

Robust cell polarity is a dynamic state established by coupling transport and GTPase signaling

Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner. Here, we investigate the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity. Using analysis of fluorescence recovery after photobleaching, we found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle. A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast. Inhibition of actin-based transport resulted in unstable Cdc42 polar caps. Unstable polarity was also observed in mutants lacking Bem1, a protein previously implicated in a feedback loop for Cdc42 activation through a signaling pathway. When Bem1 and actin were both inhibited, polarization completely failed. These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux.     

The mammalian septin MSF localizes with microtubules and is required for completion of cytokinesis

Cytokinesis in animal cells involves the contraction of an actomyosin ring formed at the cleavage furrow. Nuclear division, or karyokinesis, must be precisely timed to occur before cytokinesis in order to prevent genetic anomalies that would result in either cell death or uncontrolled cell division. The septin family of GTPase proteins has been shown to be important for cytokinesis although little is known about their role during this process. Here we investigate the distribution and function of the mammalian septin MSF. We show that during interphase, MSF colocalizes with actin, microtubules, and another mammalian septin, Nedd5, and coprecipitates with six septin proteins. In addition, transfections of various MSF isoforms reveal that MSF-A specifically localizes with microtubules and that this localization is disrupted by nocodazole treatment. Furthermore, MSF isoforms localize primarily with tubulin at the central spindle during mitosis, whereas Nedd5 is mainly associated with actin. Microinjection of affinity-purified anti-MSF antibodies into synchronized cells, or depletion of MSF by small interfering RNAs, results in the accumulation of binucleated cells and in cells that have arrested during cytokinesis. These results reveal that MSF is required for the completion of cytokinesis and suggest a role that is distinct from that of Nedd5.     

Evolution of chloroplast vesicle transport

Vesicle traffic plays a central role in eukaryotic transport. The presence of a vesicle transport system inside chloroplasts of spermatophytes raises the question of its phylogenetic origin. To elucidate the evolution of this transport system we analyzed organisms belonging to different lineages that arose from the first photosynthetic eukaryote, i.e. glaucocystophytes, chlorophytes, rhodophytes, and charophytes/embryophytes. Intriguingly, vesicle transport is not apparent in any group other than embryophytes. The transfer of this eukaryotic-type vesicle transport system from the cytosol into the chloroplast thus seems a late evolutionary development that was acquired by land plants in order to adapt to new environmental challenges.     

Structural polarity and directional growth of microtubules of Chlamydomonas flagella

A basic question concerning microtubule assembly is the polarity of growth, namely, whether subunits can add to either end of a growing microtubule or whether growth proceeds by subunit addition to only one end. To approach this question in an in vitro system, experiments were carried out on the addition of microtubule subunits to isolated flagellar axonemes. Flagella were detached from Chlamydomonas by brief treatment with non-ionic detergent, isolated by differential centrifugation, and incubated with crude high-speed extracts of porcine brain tissue or with purified tubulin (obtained by repetitive temperature-dependent assembly and disassembly). Electron microscopy of negatively stained samples showed as many as 11 long microtubules added at one end of more than 90% of the axonemes. Colchicine (100 μm), CaCl₂ (2.5 mm), and low temperature (0 °C) both prevented and reversed microtubule assembly but had no effect on axonemal length. In crude extracts microtubules formed on both members of the axonemal central pair but on only the A-tubule of the outer doublets. Flagellar fragments, produced by mechanical shearing, were also incubated with microtubule subunit. Single tubules formed at only one end of outer doublet fragments; the appearance of single tubules on one or both members of central pair fragments was predominantly unidirectional. Structural analysis of frayed axonemes and the asymmetry of side-arm attachments permitted the absolute polarity of the axonemal fragments to be determined and revealed that assembly proceeded by addition of subunits to the distal ends of the axonemal microtubules. Using purified brain tubulin, a limited extent of proximal addition and growth on the B-tubule also occurred. The extent of proximal addition increased with increasing protein concentration and temperature. We conclude that the microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth.     

Revisiting the role of microtubules in C. elegans polarity

Cells must break symmetry to acquire polarity. Microtubules have been implicated in the induction of asymmetry in several cell types, but their role in the Caenorhabditis elegans zygote, a classic polarity model, has remained uncertain. One study (see Tsai and Ahringer on p. 397 of this issue) brings new light to this problem by demonstrating that severe loss of microtubules impairs polarity onset in C. elegans.     

Single vesicle analysis of endocytic fission on microtubules in vitro

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.     

Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior

The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. Here we analyze the function of the different PAR-1 isoforms and find that only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. Our results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte.