Polarized growth in the absence of F-actin in Saccharomyces cerevisiae exiting quiescence

BACKGROUND: Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging. PRINCIPAL FINDINGS: We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis

Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path.     

Transcytosis of pancreatic bile salt-dependent lipase through human Int407 intestinal cells.

In previous studies, we have shown that the bile-salt-dependent-lipase (BSDL), secreted by pancreatic acinar cells and secreted into the duodenal lumen, can be transcytosed through intestinal cells up to the lamina propria. In this study, we used an in vitro system to provide insights into the apical to basolateral transport of BSDL, across the intestinal barrier. The Int407 human epithelial cell line, grown under conditions that optimize polarity, was used as a tight epithelium model. We attempted to delineate uptake mechanisms and the transcytotic pathway followed by this pancreatic enzyme within the intestinal Int407 cells, which do not produce BSDL. When added to the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first interact with the apical membrane. Further, BSDL forms clusters that are internalized via clathrin-coated pits. Following endocytosis, BSDL is directed to a nocodazole- and colchicin-sensitive multivesicular compartment. Interestingly, this protein transits through the Golgi apparatus, where it was found to colocalize with the KDEL retrieval-receptor. Finally, enzymatically active intact BSDL was released at the basolateral membrane level. This is the first demonstration for an apical-to-basolateral transcytotic pathway of a secreted pancreatic digestive enzyme through polarized intestinal cells.     

Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.     

Cell polarity emerges at first cleavage in sea urchin embryos

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G_M1, integrins, and the aPKC-PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical-basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G_M1, integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.     

Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans

We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans.     

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. Here we show that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell intercalation defects. We delineate a pathway that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, we provide evidence that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin.     

Cell polarity in myelinating glia: from membrane flow to diffusion barriers

Myelin-forming glia are highly polarized cells that synthesize as an extension of their plasma membrane, a multilayered myelin membrane sheath, with a unique protein and lipid composition. In most cells polarity is established by the polarized exocytosis of membrane vesicles to the distinct plasma membrane domains. Since myelin is composed of a stack of tightly packed membrane layers that do not leave sufficient space for the vesicular trafficking, we hypothesize that myelin does not use polarized exocytosis as a primary mechanism, but rather depends on lateral transport of membrane components in the plasma membrane. We suggest a model in which vesicle-mediated transport is confined to the cytoplasmic channels, from where transport to the compacted areas occurs by lateral flow of cargo within the plasma membrane. A diffusion barrier that is formed by MBP and the two adjacent cytoplasmic leaflets of the myelin bilayers acts a molecular sieve and regulates the flow of the components. Finally, we highlight potential mechanism that may contribute to the assembly of specific lipids within myelin. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development.     

Spatial modeling of vesicle transport and the cytoskeleton: the challenge of hitting the right road

The membrane trafficking machinery provides a transport and sorting system for many cellular proteins. We propose a mechanistic agent-based computer simulation to integrate and test the hypothesis of vesicle transport embedded into a detailed model cell. The method tracks both the number and location of the vesicles. Thus both the stochastic properties due to the low numbers and the spatial aspects are preserved. The underlying molecular interactions that control the vesicle actions are included in a multi-scale manner based on the model of Heinrich and Rapoport (2005). By adding motor proteins we can improve the recycling process of SNAREs and model cell polarization. Our model also predicts that coat molecules should have a high turnover at the compartment membranes, while the turnover of motor proteins has to be slow. The modular structure of the underlying model keeps it tractable despite the overall complexity of the vesicle system. We apply our model to receptor-mediated endocytosis and show how a polarized cytoskeleton structure leads to polarized distributions in the plasma membrane both of SNAREs and the Ste2p receptor in yeast. In addition, we can couple signal transduction and membrane trafficking steps in one simulation, which enables analyzing the effect of receptor-mediated endocytosis on signaling.     

Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling

Many cells show a polarized distribution of some plasma membrane proteins, which may be maintained either by a diffusion barrier or kinetically: as first demonstrated in fibroblasts, locally exocytosed proteins will remain polarized if they are endocytosed and recycled before they can diffuse to equilibrium. In yeast, actin cables direct exocytosis to the bud and to the tips of polarized mating intermediates termed shmoos. A septin ring at the bud neck retains some proteins, but shmoos lack this. Here, we show that the exocytic SNARE Snc1 is kinetically polarized. It is concentrated at bud and shmoo tips, and this requires its endocytosis. Kinetic polarization is possible in these small cells because proteins diffuse much more slowly in the yeast plasma membrane than would be expected from measurements in animal cells. Slow diffusion requires neither the cell wall nor polymerized actin, but it is affected in the ergosterol synthesis mutant erg6. Other proteins also require endocytosis for efficient polarization, and the plasma membrane SNARE Sso1 can be polarized merely by appending an endocytic signal. Thus, despite their small size, yeast cells can use localized exocytosis and endocytic recycling as a simple mechanism to maintain polarity.     

A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

Increased intracellular H(+) efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1(-)) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1(-) cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI((3,4,5))P(3)). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI((3,4,5))P(3) production and for attaining a polarized phenotype.     

Cdc42 localization and cell polarity depend on membrane traffic

Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6-aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.     

The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans

Filamentous fungi are ideal systems to study the process of polarized growth, as their life cycle is dominated by hyphal growth exclusively at the cell apex. The actin cytoskeleton plays an important role in this growth. Until now, there have been no tools to visualize actin or the actin-binding protein fimbrin in live cells of a filamentous fungus. We investigated the roles of actin (ActA) and fimbrin (FimA) in hyphal growth in Aspergillus nidulans . We examined the localization of ActA::GFP and FimA::GFP in live cells, and each displayed a similar localization pattern. In actively growing hyphae, cortical ActA::GFP and FimA::GFP patches were highly mobile throughout the hypha and were concentrated near hyphal apices. A patch-depleted zone occupied the apical 0.5 μm of growing hypha. Both FimA::GFP and Act::GFP also localize transiently to septa. Movement and later localization of both was compromised after cytochalasin treatment. Disruption of fimA resulted in delayed polarity establishment during conidium germination, abnormal hyphal growth and endocytosis defects in apolar cells. Endocytosis was severely impaired in apolar fimA disruption cells. Our data support a novel apical recycling model which indicates a critical role for actin patch-mediated endocytosis to maintain polarized growth at the apex.     

Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 +/- 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.     

Calmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway

Membrane polarity is maintained by a complex intermingling of various trafficking pathways, including basolateral and apical endocytosis. The present work was undertaken to better define the role of basolateral endocytic transport in apical membrane homeostasis. When polarized HepG2 hepatoma cells were incubated with calmodulin antagonists, the cells lost their polarity, as reflected by an inhibition of lipid transport of a fluorescent sphingomyelin to the apical membrane and an impediment of its recycling to the basolateral membrane. Instead, an accumulation of the lipid in dilated early endosomal compartments was observed, presumably due to a frustration of vesiculation. Interestingly, lipid transport to the apical pole, lipid recycling to the basolateral membrane and cell polarity were reestablished, while dilated compartments disappeared, when the cells were simultaneously treated with specific inhibitors of protein kinase C (PKC). Consistently, following activation of PKC, extensive dilation/vacuolation of early sorting endosomes was observed, very similar as seen upon treatment with calmodulin antagonists. Thus, the results indicate that membrane trafficking at early steps of the basolateral endocytic pathway in HepG2 cells is regulated by an intricate interplay between calmodulin and PKC. This interference, although not affecting endocytosis as such, compromises cell polarity by impeding membrane trafficking from early endosomes to the apical membrane.     

Recycling,clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.