Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasma membrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24p, the GEF for the rho family GTPase, Cdc42p. In its GTP-bound form, Cdc42p activates a multiprotein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre, and it has been postulated that vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research.     

In Candida albicans hyphae,Sec2p is physically associated with SEC2 mRNA on secretory vesicles

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans‐Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.     

The Rab GTPase YPT‐1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa

Vesicle traffic involves budding, transport, tethering and fusion of vesicles with acceptor membranes. GTP‐bound small Rab GTPases interact with the membrane of vesicles, promoting their association with other factors before their subsequent fusion. Filamentous fungi contain at their hyphal apex the Spitzenkörper (Spk), a multivesicular structure to which vesicles concentrate before being redirected to specific cell sites. The regulatory mechanisms ensuring the directionality of the vesicles that travel to the Spk are still unknown. Hence, we analyzed YPT‐1, the Neurospora crassa homologue of Saccharomyces cerevisiae Ypt1p (Rab1), which regulates different secretory pathway events. Laser scanning confocal microscopy revealed fluorescently tagged YPT‐1 at the Spk and putative Golgi cisternae. Co‐expression of YPT‐1 and predicted post‐Golgi Rab GTPases showed YPT‐1 confined to the Spk microvesicular core, while SEC‐4 (Rab8) and YPT‐31 (Rab11) occupied the Spk macrovesicular peripheral layer, suggesting that trafficking and organization of macro and microvesicles at the Spk are regulated by distinct Rabs. Partial colocalization of YPT‐1 with USO‐1 (p115) and SEC‐7 indicated the additional participation of YPT‐1 at early and late Golgi trafficking steps.     

Golgi organization and the apical extension of fungal hyphae: an essential relationship

The Golgi apparatus performs crucial functions in the sorting and processing of proteins destined for secretion from eukaryotic cells. In filamentous fungi, organization of the Golgi apparatus reflects the unique challenges brought about by the highly polarized nature of hyphal growth. Recent results show that Golgi compartments are spatially segregated within hyphal tip cells in a manner that depends upon the integrity of the cytoskeleton. Moreover, loss of normal Golgi organization stops polarized hyphal extension and triggers de‐polarization of the hyphal tip. These results emphasize the point that a spatially organized and dynamic Golgi apparatus represents an adaptation that is as important for hyphal extension as is the presence of a Spitzenkörper. In addition, they also identify regulatory mechanisms that could enable controlled de‐polarization of hyphae during development or infection‐related morphogenesis.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip‐growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long‐range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three‐dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub‐apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short‐range “on‐site” secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.     

Evidence That Spitzenkörper Behavior Determines the Shape of a Fungal Hypha: A Test of the Hyphoid Model

Bartnicki-Garcia, S. Bartnicki, D. D., Gierz, G., López-Franco, R., and Bracker, C. E. 1995. Evidence that Spitzenkörper behavior determines the shape of a fungal hypha; A test of the hyphoid model. Experimental Mycology 19, 153-159. Hyphae of the fungus Rhizoctonia solani have a characteristic Spitzenkörper in their growing tips and a cell shape described by the mathematical hyphoid equation. A mild disturbance of hyphae growing in a slide culture chamber on a microscope stage caused the Spitzenkörper to move away from its usual position next to the apical pole and wander briefly inside the apical dome. Hyphal elongation rate declined abruptly, and the apex became rounded and increased in diameter. As the Spitzenkörper migrated back to its polar position, rapid cell elongation resumed, and the contour of the growing hyphal tip returned to the typical hyphoid shape. The brief dislocation of the Spitzenkörper left a permanent bulge in the hyphal profile. This morphogenetic sequence was mimicked by computer simulation, based on the hyphoid equation which relates the generation of hyphal shape to the linear displacement of a vesicle supply center (VSC). The VSC was programmed to retrace the observed movements of the Spitzenkörper during the above sequence. The resulting similarity of shape between real and computer-simulated cells reinforces the mathematical prediction that the Spitzenkörper acts as a VSC and that its continuous linear advancement generates a typical hyphal tube with the characteristic hyphoid shape. Accordingly, the hyphoid model and its VSC concept provide a plausible hypothesis to explain the cellular basis of polarized growth of fungal hyphae.     

Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase,DnfB

Endocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P‐Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P‐type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact‐C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4‐ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo.     

Ypt32 recruits the Sec4p guanine nucleotide exchange factor,Sec2p,to secretory vesicles; evidence for a Rab cascade in yeast

SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.     

Functional stratification of the Spitzenkörper of Neurospora crassa

GS‐1 (ncu04189) is a protein required for the synthesis of β‐1,3‐glucan in Neurospora crassa. As chitin, β‐1,3‐glucan is a morphogenetically relevant component of the fungal cell wall. Previously, we showed that chitin synthases are delivered to the growing hyphal tip of N. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the Spitzenkörper (Spk). Tagged with the green fluorescent protein (GFP), GS‐1 accumulated in the hyphal apex forming a dynamic and pleomorphic ring‐like structure (‘Spitzenring’) that corresponded to the Spk outer macrovesicular stratum and surrounded the inner core of chitin synthase‐containing microvesicles. TIRF microscopy revealed that GS‐1‐GFP reached the hyphal apex as a population of heterogeneous‐size particles that moved along defined paths. On sucrose density gradients, GS‐1‐associated particles mainly sedimented in a high density range 1.1272–1.2124 g ml^?1. Clearly, GS‐1 and chitin synthases of N. crassa are contained in two different types of secretory vesicles that accumulate in different strata of the Spk, a differentiation presumably related to the spatial control of cell‐wall synthesis.     

Characterization of Aspergillus nidulans RabC/Rab6

The Aspergillus nidulans Golgi is not stacked. Early and late Golgi equivalents (GEs) are intermingled but can be resolved by epifluorescence microscopy. RabC, the Aspergillus ortholog of mammalian Rab6, is present across the Golgi, preferentially associated with early GEs near the tip and with late GEs in tip‐distal regions. rabCΔ mutants, showing markedly impaired apical extension, have conspicuously fragmented, brefeldin A‐insensitive early and late GEs, indicating that the Golgi network organization requires RabC. rabCΔ Golgi fragmentation is paralleled by an increase in early endosome abundance. rabCΔ reduces extracellular levels of the major secretable protease, suggesting that it impairs secretion. Notably, the Spitzenkörper, an apical intracellular structure in which secretory carriers accumulate awaiting fusion with the adjacent plasma membrane (PM), contains RabC. rabCΔ leads to abnormally increased accumulation of carriers, detectable with secretory v‐SNARE GFP‐SynA and FM4‐64, in this structure. VpsT^Vps10, present across the Golgi, recycles between endosomes and Golgi and is mislocalized to a cytosolic haze by rabCΔ that, in contrast, does not affect SynA recycling between endosomes and the PM, indicating that SynA follows a RabC‐independent pathway. tlg2Δ mutants grow normally but are synthetically lethal with rabCΔ, indicating that RabC plays Tlg2‐independent roles.     

Spitzenk?rper,Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.Polarized growth of fungi requires that a supply of secretory vesicles is delivered along cytoskeletal tracks to the site of cell expansion (for reviews, see references 13, 29, 30, and 31). Fusion of these membrane-bound vesicles with the plasma membrane allows the necessary expansion of the plasma membrane and releases the enzymes and raw materials for the synthesis of new cell wall material and the remodeling necessary to allow this newly synthesized material to be inserted into the existing cell wall. The process of polarized growth has been extensively studied in the budding yeast Saccharomyces cerevisiae and provides a model for studying the process in other fungi (for a review, see reference 20). Post-Golgi vesicles travel to sites of polarized growth along actin cables (23). Actin cables are nucleated at sites of polarized growth by the formin Bni1 facilitated by a multiprotein complex called the polarisome, which consists of Spa2, Bud6, and Pea1(5, 22, 24, 27). The motive force for vesicle transport is provided by Myo2, a class V myosin, complexed to its regulatory light chain Mlc1 (22, 26). At the plasma membrane, secretory vesicles dock with a second multiprotein complex called the exocyst before fusion with the plasma membrane (14, 15, 32, 33), mediated by v-SNARES on the vesicle and t-SNARES on the membrane. The exocyst is an octomeric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (21). It is thought that Sec3 and a fraction of the Exo70 pool are localized at sites of polarized growth independently of the actin cytoskeleton (3, 6). The other exocyst subunits and the remainder of the Exo70 pool are thought to be transported to sites of polarized growth on secretory vesicles, where together with Sec3 and Exo70 they form the exocyst complex (3). Secretory vesicles exit the Golgi apparatus, travel toward sites of polarized growth, and dock with the exocyst by use of the Rab-type GTPase Sec4 in its GTP-bound form, which is activated by its GEF, Sec2 (12, 19, 35, 36). In the S. cerevisiae cell cycle, polarized growth is initially directed toward the bud tip in young buds (17). Growth subsequently becomes isotropic in larger buds before being directed toward the mother bud neck during cytokinesis at the end of the cell cycle. Accordingly, polarisome and exocyst components localize to the tips of young buds (7, 27, 28).The rate of hyphal tip extension is much greater than that of the growth of a yeast or pseudohyphal bud. In rich yeast extract-peptone-dextrose (YEPD) medium, Candida albicans hyphae extend at the rate of 0.25 μm min⁻¹, compared to 0.0625 μm min⁻¹ in yeast buds and 0.125 μm min⁻¹ in pseudohyphal cells (P. Sudbery unpublished observations). In hyphae of filamentous fungi, a structure called a Spitzenkörper is present at the tip, which is rich in secretory vesicles (8, 9, 11, 29, 34). It is believed that the Spitzenkörper acts as a vesicle supply center (VSC) (1). This model proposes that the Spitzenkörper is maintained at a fixed distance from the hyphal tip. Vesicles radiate out in equal directions to fuse with the plasma membrane, so that more vesicles per unit area fuse with the hyphal tip itself than with other parts of the hyphae. Mathematical modeling shows that this explains the distinctive shape of hyphal tips.In order to investigate the mechanism of polarized growth in the hyphae of Candida albicans, we previously determined the localization of Mlc1-yellow fluorescent protein (YFP) and the polarisome components Bud6-YFP and Spa2-YFP (4). We found that in hyphae, polarisome components localized to a surface crescent, as they did in young yeast buds and the tips of elongated pseudohyphal buds. However, in hyphae Mlc1-YFP localized to a bright spot, which at least in some hyphae was clearly inside the tip, rather than at the surface, and which appeared spherical in three-dimensional reconstructions. We concluded that this represented a Spitzenkörper. In some hyphae Mlc1-YFP also localized to a surface crescent, similar to the pattern displayed by polarisome components. This observation suggested that the Spitzenkörper and polarisome were separate structures, both of which were present at hyphal tips, but that only the polarisome was present at the bud tips of pseudohyphae and yeast. Moreover, the dual localization of Mlc1-YFP to a crescent and a spot suggested that Mlc1 may be present in both structures.While S. cerevisiae has proved to be an excellent model to investigate the molecular genetics of polarized growth, it is less optimal to study the spatial organization of the molecular components because polarized growth of the bud is restricted to a short period after bud emergence when the nascent bud is small. Thus, there has been little effort to investigate the fine detail of the spatial organization of the different components of the polarization machinery beyond noting that they localize to sites of polarized growth. In this study we exploited the opportunities afforded by the continuous polarized growth of C. albicans hyphae to clarify the relationship between the Spitzenkörper, polarisome, and exocyst, which cooperate to mediate the extreme polarized growth of hyphae. We show that the vesicle-associated marker Sec4 also localizes to a Spitzenkörper-like structure, confirming the existence of a vesicle-rich area corresponding to a Spitzenkörper at the hyphal tip. We show that exocyst components such as Sec3, Sec6, Sec8, Exo70, and Exo84 localize to a surface crescent, so the exocyst, like the polarisome, is also a spatially separate structure from the Spitzenkörper. We used three independent strategies to investigate the dynamic properties of these structures. Fluorescence recovery after photobleaching (FRAP) was used to measure the rate at which new proteins arrived at the tip. Fluorescence loss in photobleaching (FLIP) was used to measure the rate at which proteins exited the tip. Cytochalasin A was used to disrupt actin cables, allowing the persistence of proteins at the tip to be measured after the supply of new proteins was blocked. In each case we found that Spitzenkörper components Sec4, Sec2, and Mlc1 were highly dynamic, while the polarisome component Spa2 was stable. Intriguingly, exocyst components showed intermediate dynamic properties, suggesting that they are delivered to the tip on vesicles but that not all vesicles carry a complement of exocyst components. We suggest that these data are consistent with a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.     

The Sec34/Sec35p complex,a Ypt1p effector required for retrograde intra-Golgi trafficking,interacts with Golgi SNAREs and COPI vesicle coat proteins

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.     

Contact‐induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano‐fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour‐following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ～ 8.7 μN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.     

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.     

Vesicle trafficking via the Spitzenkörper during hyphal tip growth in Rhizoctonia solani

Growing hyphae of Rhizoctonia solani were stained with the endocytic marker dye FM4-64 and imaged by confocal microscopy. Staining of the plasma membrane was followed by labeling of organelles in the cytoplasm (after ~1 min) and of the Spitzenkörper (Spk; after ~2 min). Fluorescence recovery after photobleaching (FRAP) of the stained Spk demonstrated the vectorial flow of secretory vesicles from the apical cytoplasm to the Spk. This flux was modelled in a two-compartment model. The turnover time of the vesicles of the Spk was estimated to be 1.3–2.5 min. These results are roughly consistent with the expected flux of vesicles through the Spk based on the number of secretory vesicles within the Spk and the number of secretory vesicles that would be necessary to fuse with the apical plasma membrane to maintain hyphal extension rates. These results suggest that membrane retrieval via endocytosis is not as significant as previously suggested.     

Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae . We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro . In vivo , both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.     

Segregation of the Qb‐SNAREs GS27 and GS28 into Golgi Vesicles Regulates Intra‐Golgi Transport

The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi‐resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb‐SNAREs GS27 (membrin) and GS28 (GOS‐28), and depleted of nucleotide sugar transporters. A block of intra‐Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra‐Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP‐galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra‐Golgi transport; re‐addition of isolated Golgi vesicles devoid of UDP‐galactose translocase obtained from normal cells restores intra‐Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter‐cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis‐to‐trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra‐Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter‐cisternal connections .