Polarization Of Actin Cytoskeleton Research Articles

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

E‐Cigarette Use Increases Susceptibility to Bacterial Infection by Impairment of Human Neutrophil Chemotaxis, Phagocytosis and NET Formation

Although E‐cigarettes are frequently portrayed as a safer alternative to conventional cigarettes, a growing body of evidence suggests that the use of these devices may adversely affect host defense against infection. Neutrophils, critical effector cells of the innate immune system and early responders to sites of infection, may represent a particularly important cellular target of E‐cigarette vapor components. Using primary human neutrophils isolated from healthy donors, we evaluated the impact of electronic vapor extract (EVE) exposure on multiple aspects of neutrophil cellular physiology, including chemotaxis, reactive oxygen species (ROS) production, generation of Neutrophil Extracellular Traps (NETs) and phagocytosis. Using a transwell chamber approach, we found that exposure to E‐cigarette vapor‐infused media (EVE) reduced neutrophil chemotaxis in response to the chemoattractant f‐Met‐Leu‐Phe (fMLP) by up to 4.2 fold (p < 0.001). Staining with the fluorescent actin probe rhodamine phalloidin revealed aberrant actin cytoskeleton polarization in EVE‐treated, fMLP‐stimulated neutrophils. Neutrophil membrane fluidity was also altered by EVE exposure, with holotomographic microscopy revealing alterations in cellular morphology in EVE‐treated neutrophils. ROS production was reduced in EVE‐treated neutrophils, and ROS‐dependent production of bactericidal extracellular traps was reduced by up to 3.5 fold (p < 0.01). Interestingly, this reduction in NET production (NETosis) was observed in neutrophils treated with EVE produced using propylene glycol‐based E‐cigarette liquid formulations both with and without nicotine. The rate of neutrophil phagocytosis, quantified using fluorescent E. coli/S. aureus bioparticles, was also reduced by up to 47% in EVE‐treated neutrophils (p < 0.05). To determine how such alterations of immune cell function might affect host response to infection, a physiologic mouse model of chronic E‐cigarette exposure and sepsis was utilized. In this model, E‐cigarette vapor inhalation led to reduced neutrophil migration into infected spaces and a higher burden of Pseudomonas aeruginosa. Taken together, these findings provide evidence that E‐cigarette use adversely impacts the innate immune system, potentially placing E‐cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.

α-parvin is required for epidermal morphogenesis, hair follicle development and basal keratinocyte polarity.

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.

Differences in Three-Dimensional Geometric Recognition by Non-Cancerous and Cancerous Epithelial Cells on Microgroove-Based Topography

During metastasis, cancer cells are exposed to various three-dimensional microstructures within the body, but the relationship between cancer migration and three-dimensional geometry remain largely unclear. Here, such geometric effects on cancerous cells were investigated by characterizing the motility of various cancer cell types on microgroove-based topographies made of polydimethylsiloxane (PDMS), with particular emphasis on distinguishing cancerous and non-cancerous epithelial cells, as well as understanding the underlying mechanism behind such differences. The 90-degree walls enhanced motility for all cell lines, but the degrees of enhancements were less pronounced for the cancerous cells. Interestingly, while the non-cancerous epithelial cell types conformed to the three-dimensional geometrical cues and migrated along the walls, the cancerous cell types exhibited a unique behavior of climbing upright walls, and this was associated with the inability to form stable, polarized actin cytoskeleton along the walls of the microgrooves. Furthermore, when non-cancerous epithelial cell lines were altered to different levels of polarization capabilities and cancer malignancy or treated with inhibitory drugs, their three-dimensional geometry-dependent motility approached those of cancerous cell lines. Overall, the results suggest that cancerous cells may gradually lose geometrical recognition with increasing cancer malignancy, allowing them to roam freely ignoring three-dimensional geometrical cues during metastasis.

Apical Sarcomere-like Actomyosin Contracts Nonmuscle Drosophila Epithelial Cells

Actomyosin networks generate contractile force that changes cell and tissue shape. In muscle cells, actin filaments and myosin II appear in a polarized structure called a sarcomere, in which myosin II is localizedin the center. Nonmuscle cortical actomyosin networks are thought to contract when nonmuscle myosin II (myosin) is activated throughout a mixed-polarity actin network. Here, we identified a mutant version of the myosin-activating kinase, ROCK, that localizes diffusely, rather than centrally, in epithelial cell apices. Surprisingly, this mutant inhibits constriction, suggesting that centrally localized apical ROCK/myosin activity promotes contraction. We determined actin cytoskeletal polarity by developing a barbed end incorporation assay for Drosophila embryos, which revealed barbed end enrichment at junctions. Our results demonstrate that epithelial cells contract with a spatially organized apical actomyosin cortex, involving a polarized actin cytoskeleton and centrally positioned myosin, with cell-scale order that resembles a muscle sarcomere.

Front-Rear Polarization by Mechanical Cues: From Single Cells to Tissues.

Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms.

Role of heparan sulfate in mediating CXCL8-induced endothelial cell migration.

CXCL8 (Interleukin-8, IL-8) plays an important role in angiogenesis and wound healing by prompting endothelial cell migration. It has been suggested that heparan sulfate (HS) could provide binding sites on endothelial cells to retain and activate highly diffusible cytokines and inflammatory chemokines. In the present study, we aimed to test the hypothesis that HS is essential for enhancement of endothelial cell migration by CXCL8, and to explore the underlying mechanism by detecting the changes in expression and activity of Rho GTPases and in the organization of actin cytoskeleton after enzymatic removal of HS on human umbilical vein endothelial cells (HUVECs) by using heparinase III. Our results revealed that the wound healing induced by CXCL8 was greatly attenuated by removal of HS. The CXCL8-upregulated Rho GTPases including Cdc42, Rac1, and RhoA, and CXCL8-increased Rac1/Rho activity were suppressed by removal of HS. The polymerization and polarization of actin cytoskeleton, and the increasing of stress fibers induced by CXCL8 were also abolished by heparinase III. Taken together, our results demonstrated an essential role of HS in mediating CXCL8-induced endothelial cell migration, and highlighted the biological importance of the interaction between CXCL8 and heparan sulfate in wound healing.

The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology-Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 "restrains" the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.

Polarization of actin cytoskeleton is reduced in dendritic protrusions during early spine development in hippocampal neuron

Dendritic spines are small protrusions that receive synaptic signals in neuronal networks. The actin cytoskeleton plays a key role in regulating spine morphogenesis, as well as in the function of synapses. Here we report the first quantitative measurement of F-actin retrograde flow rate in dendritic filopodia, the precursor of dendritic spines, and in newly formed spines, using a technique based on photoactivation localization microscopy. We found a fast F-actin retrograde flow in the dendritic filopodia but not in the spine necks. The quantification of F-actin flow rates, combined with fluorescence recovery after photobleaching measurements, allowed for a full quantification of spatially resolved kinetic rates of actin turnover, which was not previously feasible. Furthermore we provide evidences that myosin II regulates the actin flow in dendritic filopodia and translocates from the base to the tip of the protrusion upon spine formation. Rac1 inhibition led to mislocalization of myosin II, as well as to disruption of the F-actin flow. These results provide advances in the quantitative understanding of F-actin remodeling during spine formation.

Structural Analyses of the Slm1-PH Domain Demonstrate Ligand Binding in the Non-Canonical Site

BackgroundPleckstrin homology (PH) domains are common membrane-targeting modules and their best characterized ligands are a set of important signaling lipids that include phosphatidylinositol phosphates (PtdInsPs). PH domains recognize PtdInsPs through two distinct mechanisms that use different binding pockets on opposite sides of the β-strands 1 and 2: i) a canonical binding site delimited by the β1-β2 and β3-β4loops and ii) a non-canonical binding site bordered by the β1-β2 and β5-β6loops. The PH domain-containing protein Slm1 from budding yeast Saccharomyces cerevisiae is required for actin cytoskeleton polarization and cell growth. We recently reported that this PH domain binds PtdInsPs and phosphorylated sphingolipids in a cooperative manner.Principal FindingsTo study the structural basis for the Slm1-PH domain (Slm1-PH) specificity, we co-crystallized this domain with different soluble compounds that have structures analogous to anionic lipid head groups of reported Slm1 ligands: inositol 4-phosphate, which mimics phosphatidylinositol-4-phosphate (PtdIns(4)P), and phosphoserine as a surrogate for dihydrosphingosine 1-phosphate (DHS1-P). We found electron densities for the ligands within the so-called non-canonical binding site. An additional positively charged surface that contacts a phosphate group was identified next to the canonical binding site.ConclusionsOur results suggest that Slm1-PH utilizes a non-canonical binding site to bind PtdInsPs, similar to that described for the PH domains of β-spectrin, Tiam1 and ArhGAP9. Additionally, Slm1-PH may have retained an active canonical site. We propose that the presence of both a canonical and a non-canonical binding pocket in Slm1-PH may account for the cooperative binding to PtdInsPs and DHS-1P.

Tor Complex 1 Controls Telomere Length by Affecting the Level of Ku

Telomeres are specialized DNA-protein structures at the ends of eukaryotic chromosomes. Telomeric DNA is synthesized by telomerase, which is expressed only at the early stages of development [1, 2]. To become malignant, any cell has to be able to replenish telomeres [3]. Thus, understanding how telomere length is monitored has significant medical implications, especially in the fields of aging and cancer. In yeast, telomerase is constitutively active. A large network of genes participates in controlling telomere length [4-8]. Tor1 and Tor2 (targets of rapamycin [9]) are two similar kinases that regulate cell growth [10]. Both can be found as part of the TOR complex 1 (TORC1 [11]), which coordinates the response to nutrient starvation and is sensitive to rapamycin [12]. The rapamycin-insensitive TOR complex 2 (TORC2) contains only Tor2 and regulates actin cytoskeleton polarization [13]. Here we provide evidence for a role of TORC1 in telomere shortening upon starvation in yeast cells. The TORC1 signal is transduced by the Gln3/Gat1/Ure2 pathway, which controls the levels of the Ku heterodimer, a telomere regulator. We discuss the potential implications for the usage of rapamycin as a therapeutic agent against cancer and the effect that calorie restriction may have on telomere length.

PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9

The mesenchymal mode of cancer cell invasion characterized by active adhesion turnover and a polarized actin cytoskeleton, is critically regulated by the adaptor protein NEDD9/HEF1/Cas-L. While it is known that NEDD9 is subject to extensive phosphorylation modification, the molecules that determine NEDD9 phosphorylation to stimulate adhesion turnover and mesenchymal cell morphologies are currently unknown. Earlier studies have suggested that the serine/threonine phosphatase PP2A regulates interconversion between a low molecular mass NEDD9 phosphoform and higher molecular mass phosphoforms. However, previous studies have used chemical inhibitors to block PP2A activity. In the present study we therefore aimed to specifically inhibit PP2A activity via siRNA and dominant negative approaches to investigate the effect of PP2A on interconversion between 115kDa and 105kDa NEDD9 and determine the functional consequence of PP2A activity for NEDD9 function. Strikingly, we find that while the phosphatase inhibitor Calyculin A indeed abrogates detachment-induced dephosphorylation of the 115kDa NEDD9 phosphoform, PP2A depletion does not inhibit 115kDa to 105kDa interconversion. Our data suggest instead that PP2A targets discrete NEDD9 phosphorylation modifications separate to the events that mediate interconversion between the two forms. Functionally, PP2A depletion increases NEDD9 mediated cell spreading and mutation of S369 in the serine-rich region of NEDD9 to aspartate mimics this effect. Importantly, mutation of S369 to alanine abrogates the ability of dominant negative PP2A to increase NEDD9-mediated cell spreading. Collectively, our data reveal that the tumour suppressor PP2A may act via S369 to regulated NEDD9-mediated cell spreading.

Functional analysis of Candida albicans genes encoding SH3-domain-containing proteins

Postgenomic gene-function analyses with Candida albicans are hindered by its constitutive diploidy and the lack of a sexual cycle. Rapid generation of mutant strains can be achieved using PCR-based techniques for directed gene alterations. Here, we report the analyses of nine C. albicans genes that encode Src Homology 3-domain proteins. Phenotypic analyses included the potential of the mutants to form hyphal filaments, maintain a polarized actin cytoskeleton or the ability to generate large vacuoles in the germ cells and in subapical compartments. The C. albicans homologs of the Saccharomyces cerevisiae BBC1, BOI2, BUD14, FUS1, HSE1, PIN3, RVS167, RVS167-2 and SHO1 were all found to be nonessential. Deletion of RVS167 resulted in a strain with a decreased ability to form hyphal filaments. The number of cortical actin patches was increased in Deltarvs167 strains and their distribution was depolarized in both mother and daughter yeast cells and along the hyphae during filamentous growth stages. Polarization of patches could be restored upon reintroduction of the wild-type gene. Deletion of BOI2 was found to generate a defect in vacuolar fusion in hyphae. In contrast to a deletion in the Deltawal1 gene, Deltaboi2 cells formed abundant hyphae, indicating that fragmented vacuoles do not inhibit filamentation. Placing BOI2 under control of the MAL2-promoter allowed the regulation of this phenotype depending on the growth conditions.

Loss of Fyn kinase Impairs Oocyte Quality and Developmental Potential.

Fyn kinase is highly expressed in mammalian and non-mammalian oocytes and has been implicated in the signal transduction events including calcium signaling, cell cycle events such as meiosis resumption and pronuclear congression. The results of the above experimental studies contradict other reports that the Fyn-null mice exhibit normal fertility. The purpose of the present study was to compare the fertility of B6/129S7-Fyntm1Sor/J female mice with females from the parent strain (B6/129SF2/J) (The Jackson Laboratory, Bar Harbor, ME). Western blot analysis revealed the absence of Fyn protein in Fyn-null oocytes, although these oocytes did over express Yes protein kinase, possibly in an attempt to compensate for the loss of Fyn. Fyn-null females were smaller in size than controls and produced significantly smaller litter sizes during the first five parturitions. The interval between litters was longer and the overall reproductive lifespan was shorter than controls. Superovulation of age-matched females produced oocytes that were similar in number and size to controls, but exhibited a thinner zona pellucida and a marked accumulation of dense cytoplasmic inclusions of unknown composition. Evaluation by confocal microscopy revealed that approximately 18% of the ovulated eggs recovered from the oviduct at 15hrs post hCG administration were immature (MI) which was significantly higher than controls. Fyn-null oocytes also exhibited defects in spindle organization as well as reduced ability to position the MII spindle close to the egg cortex. These spindle abnormalities correlated with defects in cortical actin cytoskeleton polarity. Fertilization in vitro with wild type sperm revealed that the Fyn-null oocytes were fertilized at a rate similar to controls, but exhibited reduced developmental potential since 73% of fertilized eggs (pronuclei) reached the 2-cell stage vs 98% from controls. Embryos that reached the 2-cell stage developed to blastocyst at a rate similar to controls. Calcium imaging studies revealed that Fyn-null oocytes fertilized with wild type sperm exhibited reduced frequency of calcium oscillations and a modified pattern of peak amplitude, although the total calcium signal produced by Fyn-null oocytes was similar to controls. The altered calcium signaling properties were correlated with a modified distribution of IP3 receptor as detected by confocal immunofluorescence. The IP3 receptor type 1 was frequently distributed in a highly irregular pattern in Fyn-null oocytes with large clumps or accumulations that were not present in control oocytes. In summary, the Fyn-null oocyte exhibits defects in meiotic maturation, meiotic spindle integrity, cortical cytoskeleton organization, and calcium signaling that may explain the reduced developmental potential. (platform)

CAMP Inhibits Cell Migration by Interfering with Rac-induced Lamellipodium Formation

Cell migration is critical for animal development and physiological as well as pathological responses. One important step during cell migration is the formation of lamellipodia at the leading edge of migrating cells. Here we report that the second messenger cAMP inhibits the migration of mouse embryonic fibroblast cells and mouse breast tumor cells. cAMP acts downstream of the small GTPase Rac and interferes with the formation of lamellipodia. Moreover, cAMP decreases the phosphorylation of the myosin light chain at the leading edge of cells and increases the phosphorylation of the vasodilator-stimulated phosphoprotein. Together with our previous report of a positive role of another second messenger, cGMP, in lamellipodium formation, our data indicate that cAMP and cGMP play opposite roles in modulating lamellipodium formation.

Alternative Modes of Organellar Segregation during Sporulation in Saccharomyces cerevisiae

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.

Verprolin function in endocytosis and actin organization

Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.

A Conserved Docking Site Modulates Substrate Affinity for Calcineurin, Signaling Output, and In Vivo Function

Calcineurin, the conserved Ca(2+)/calmodulin-regulated protein phosphatase, mediates diverse aspects of Ca(2+)-dependent signaling. We show that substrates bind calcineurin with varying strengths and examine the impact of this affinity on signaling. We altered the calcineurin-docking site, or PxIxIT motif, in Crz1, the calcineurin-regulated transcription factor in S. cerevisiae, to decrease (Crz1(PVIAVN)) or increase (Crz1(PVIVIT)) its affinity for calcineurin. As a result, the Ca(2+)-dependent dephosphorylation and activation of Crz1(PVIAVN) are decreased, whereas Crz1(PVIVIT) is constitutively dephosphorylated and hyperactive. Surprisingly, the physiological consequences of altering calcineurin-Crz1 affinity depend on the growth conditions. Crz1(PVIVIT) improves yeast growth under several environmental stress conditions but causes a growth defect during alkaline stress, most likely by titrating calcineurin away from other substrates or regulators. Thus, calcineurin-substrate affinity determines the Ca(2+) concentration dependence and output of signaling in vivo as well as the balance between different branches of calcineurin signaling in an overall biological response.

Dynamic Organization of the Actin Cytoskeleton During Meiosis and Spore Formation in Budding Yeast

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non-polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post-meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate-rich compartments, which may be chitosome analogous structures.

Polarized growth: maintaining focus on the tip

Tip growth, a spatially focused cell expansion, has been best characterized in two plant cell types: pollen tubes and root hairs. It has long been established that both cell types require three intracellular components for this process: a tip-high calcium gradient, a polarized actin cytoskeleton, and tip-directed vesicle trafficking. More recently, additional mechanistic parallels have been observed between the two cell types, including roles for ROP and Rab GTPase signaling, phosphoinositides, calcium-dependent protein kinases, and the exocyst. Uncovering pathways that control the three components is beginning to reveal a highly interconnected network, which we call the tip growth LENS (for localization enhancing network, self-sustaining), that coordinates the required cellular activities to allow regulated tip growth, and to maintain itself as the tip advances.