Spatial guidance of cell asymmetry: septin GTPases show the way

Eukaryotic cells develop asymmetric shapes suited for specific physiological functions. Morphogenesis of polarized domains and structures requires the amplification of molecular asymmetries by scaffold proteins and regulatory feedback loops. Small monomeric GTPases signal polarity, but how their downstream effectors and targets are spatially co-ordinated to break cell symmetry is poorly understood. Septins comprise a novel family of GTPases that polymerize into non-polar filamentous structures which scaffold and restrict protein localization. Recent studies show that septins demarcate distinct plasma membrane domains and cytoskeletal tracks, enabling the formation of intracellular asymmetries. Here, we review these findings and discuss emerging mechanisms by which septins promote cell asymmetry in fungi and animals.     

septin基因家族的研究进展

septin是一个广泛存在于除植物以外所有真核生物中的基因家族。最初认为septin家族是与酵母细胞胞质分裂相关的基因家族。然而随着研究的深入, 人们发现这类基因编码的蛋白质在许多生物体内出现了较大的功能分化, 尤其在哺乳动物细胞中, 他们不仅成员众多, 且参与了细胞分裂、细胞极化、囊泡运输及胞膜重构等多个过程。更引起研究人员重视的是: 最近有大量数据表明, 这一家族的一些成员与肿瘤发生、神经功能障碍和病原微生物感染的过程直接相关。因此, 近年来septin家族的功能研究正逐步成为细胞生物学及病理学研究的新热点。文章将试图从septin基因家族的种类、结构特点、生物学功能及其与人类疾病的关系等方面的研究进展进行综述。     

Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases

To get insight into the action of Rho GTPases on the microtubule system we investigated the effects of Cdc42, Rac1, and RhoA on the dynamics of microtubules in Swiss 3T3 fibroblasts. In control cells microtubule ends were dynamic: plus ends frequently switched between growth, shortening and pauses; the growth phase predominated over shortening. Free minus ends of microtubules depolymerized rapidly and never grew. Free microtubules were short-lived, and the microtubule network was organized into a radial array. In serum-starved cells microtubule ends became more stable: although plus ends still transited between growth and shortening, polymerization and depolymerization excursions became shorter and balanced each other. Microtubule minus ends were also stabilized. Consequently lifespan of free microtubules increased and microtubule array changed its radial pattern into a random one. Activation of Cdc42 and Rac1 in serum-starved cells promoted dynamic behavior of microtubule plus and minus ends, while inhibition of these GTPases in serum-grown cells suppressed microtubule dynamics and mimicked all effects of serum starvation. Activation of RhoA in serum-grown cells had effects similar to Cdc42 /Rac1 inactivation: it suppressed the dynamics of plus and minus ends, reduced the length of growth and shrinking episodes, and disrupted the radial organization of microtubules. However, in contrast to Cdc42 and Rac1 inactivation, active RhoA had no effect on the balance between microtubule growth and shortening. We conclude that Cdc42 and Rac1 have similar stimulating effects on microtubule dynamics while RhoA acts in an opposite way.     

Rho family GTPases: more than simple switches

Rho family GTPases control a large variety of biological processes. Cycling of Rho proteins between the GDP-bound and the GTP-bound state is controlled by several classes of regulatory proteins. In this review, we discuss the signal-transduction mechanisms that control these regulators. We will emphasize the subcellular localization of Rho GTPases and their regulatory proteins and the role of GTP hydrolysis in signal transmission.     

Right place,right time - Spatial guidance of neuronal morphogenesis by septin GTPases

Neuronal morphogenesis is guided by outside-in signals and inside-out mechanisms, which require spatiotemporal precision. How the intracellular mechanisms of neuronal morphogenesis are spatiotemporally controlled is not well understood. Septins comprise a unique GTPase module, which consists of complexes with differential localizations and functions. Septins demarcate distinct membrane domains in neural precursor cells, orienting the axis of cell division and the sites of neurite formation. By controlling the localization of membrane and cytoskeletal proteins, septins promote axon-dendrite formation and polarity. Furthermore, septins modulate vesicle exocytosis at pre-synaptic terminals, and stabilize dendritic spines and post-synaptic densities in a phospho-regulatable manner. We posit that neuronal septins are topologically and functionally specialized for the spatiotemporal regulation of neuronal morphogenesis and plasticity.     

Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases

Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.     

Phosphorylation-dependent regulation of septin dynamics during the cell cycle

Septins are GTPases involved in cytokinesis. In yeast, they form a ring at the cleavage site. Using FRAP, we show that septins are mobile within the ring at bud emergence and telophase and are immobile during S, G2, and M phases. Immobilization of the septins is dependent on both Cla4, a PAK-like kinase, and Gin4, a septin-dependent kinase that can phosphorylate the septin Shs1/Sep7. Induction of septin ring dynamics in telophase is triggered by the translocation of Rts1, a kinetochore-associated regulatory subunit of PP2A phosphatase, to the bud neck and correlates with Rts1-dependent dephosphorylation of Shs1. In rts1-Delta cells, the actomyosin ring contracts properly but cytokinesis fails. Together our results implicate septins in a late step of cytokinesis and indicate that proper regulation of septin dynamics, possibly through the control of their phosphorylation state, is required for the completion of cytokinesis.     

Forchlorfenuron alters mammalian septin assembly, organization, and dynamics

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.     

Plexin signaling via off-track and rho family GTPases

Two papers in this issue of Neuron examine new aspects of Semaphorin signaling via Plexin receptors. Winberg et al. present evidence that the transmembrane protein Off-track (OTK) interacts biochemically and genetically with Plexin A and is important for Sema 1a repulsive signaling. Hu et al. examine the coupling of Plexin B to Rac and RhoA and propose that Plexin B signaling involves inhibition of Rac function by direct sequestration and simultaneous activation of RhoA.     

Phylogenetic and evolutionary analysis of the septin protein family in metazoan

Septins, a conserved family of cytoskeletal GTP-binding proteins, were presented in diverse eukaryotes. Here, a comprehensive phylogenetic and evolutionary analysis for septin proteins in metazoan was carried out. First, we demonstrated that all septin proteins in metazoan could be clustered into four subgroups, and the representative homologue of every subgroup was presented in the non-vertebrate chordate Ciona intestinalis, indicating that the emergence of the four septin subgroups should have occurred prior to divergence of vertebrates and invertebrates, and the expansion of the septin gene number in vertebrates was mainly by the duplication of pre-existing genes rather than by the appearance of new septin subgroup. Second, the direct orthologues of most human septins existed in zebrafish, which suggested that human septin gene repertoire was mainly formed by as far as before the split between fishes and land vertebrates. Third, we found that the evolutionary rate within septin family in mammalian lineage varies significantly, human SEPT1, SEPT 10, SEPT 12, and SEPT 14 displayed a relative elevated evolutionary rate compared with other septin members. Our data will provide new insights for the further function study of this protein family.     

Signaling networks linking integrins and rho family GTPases

Integrins and Rho family GTPases function coordinately to mediate adhesion-dependent events in cells. Recently, it has also become apparent that integrins regulate Rho GTPases and vice versa. Integrins and GTPases might therefore be organized into complex signaling cascades that regulate cell behavior.     

Microbial toxins and the glycosylation of rho family GTPases

Large clostridial cytotoxins act on cells by glycosylating low molecular mass GTPases using nucleotide-sugars as the sugar donor. These toxins are important virulence factors in human and animal diseases, but are also valuable cell biology tools. Recent findings shed some light on their mode of action and provide new insights into the structure/activity relationship of these bacterial toxins.     

The evolutionary dynamics of haplodiploidy: Genome architecture and haploid viability

Haplodiploid reproduction, in which males are haploid and females are diploid, is widespread among animals, yet we understand little about the forces responsible for its evolution. The current theory is that haplodiploidy has evolved through genetic conflicts, as it provides a transmission advantage to mothers. Male viability is thought to be a major limiting factor; diploid individuals tend to harbor many recessive lethal mutations. This theory predicts that the evolution of haplodiploidy is more likely in male heterogametic lineages with few chromosomes, as genes on the X chromosome are often expressed in a haploid environment, and the fewer the chromosome number, the greater the proportion of the total genome that is X‐linked. We test this prediction with comparative phylogenetic analyses of mites, among which haplodiploidy has evolved repeatedly. We recover a negative correlation between chromosome number and haplodiploidy, find evidence that low chromosome number evolved prior to haplodiploidy, and that it is unlikely that diplodiploidy has reevolved from haplodiploid lineages of mites. These results are consistent with the predicted importance of haploid male viability.     

Rho family GTPases: Making it to the third dimension

The role of Rho family GTPases in controlling the actin cytoskeleton and thereby regulating cell migration has been well studied for cells migrating on 2D surfaces. In vivo, cell migration occurs within three-dimensional matrices and along aligned collagen fibers with rather different spatial requirements. Recently, a handful of studies coupled with new approaches have demonstrated that Rho GTPases have unique regulation and roles during cell migration within 3D matrices, along collagen fibers, and in vivo. Here we propose that migration on aligned matrices facilitates spatial organization of Rho family GTPases to restrict and stabilize protrusions in the principle direction of alignment, thereby maintaining persistent migration. The result is coordinated cell movement that ultimately leads to higher rates of metastasis in vivo.     

Regulation of endocytic traffic by rho family GTPases

Endocytosis is a complicated yet highly efficient process that involves the uptake and processing of cargoes, ranging from small molecules, to activated signalling receptors, to whole microorganisms. Regulation of endocytic pathways is poorly understood. Recent evidence suggests that the Rho GTPase family of signalling proteins is intimately involved in endocytic traffic, providing novel insights into the control mechanisms that govern this process.     

Cadherin engagement regulates Rho family GTPases.

The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.