Polarized insertion of new membrane from a cytoplasmic reservoir during cleavage of the Drosophila embryo

Cellularization of the Drosophila embryo is a specialized form of cytokinesis that results in the formation of a polarized epithelium. The mechanisms of membrane growth during cytokinesis are largely unknown. It is also unclear whether membrane growth and polarization represent distinct processes that occur simultaneously or whether growth of the membrane is involved in the emergence of polarity. Using a combination of surface labeling and particles tracking techniques, we monitored the dynamics of marked membrane regions during cellularization. We find that the major source of membrane is intracellular, rather than in the form of a plasma membrane reservoir. Depolymerization of microtubules inhibits the export of a newly synthesized transmembrane protein from the Golgi apparatus to the plasma membrane and simultaneously blocks membrane growth. Membrane insertion occurs in a defined sequence at specific sites, first apical, then apical-lateral. Diffusion of the membrane appears insufficient to compete with the massive local insertion of new membrane. We thus identify a tightly regulated scheme of polarized membrane insertion during cellularization. We propose that such a mechanism could participate in the progressive emergence of apical-basal polarity.     

A link between mitotic entry and membrane growth suggests a novel model for cell size control

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.     

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background

During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.

Results

To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.

Conclusions

This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.     

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.     

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea

Increased membrane fluidity, which causes cofactor leakage and loss of membrane potential, has long been documented as a cause for decreased cell growth during exposure to ethanol, butanol, and other alcohols. Reinforcement of the membrane with more complex lipid components is thus thought to be beneficial for the generation of more tolerant organisms. In this study, organisms with more complex membranes, namely, archaea, did not maintain high growth rates upon exposure to alcohols, indicating that more complex lipids do not necessarily fortify the membrane against the fluidizing effects of alcohols. In the presence of alcohols, shifts in lipid composition to more saturated and unbranched lipids were observed in most of the organisms tested, including archaea, yeasts, and bacteria. However, these shifts did not always result in a decrease in membrane fluidity or in greater tolerance of the organism to alcohol exposure. In general, organisms tolerating the highest concentrations of alcohols maintained membrane fluidity after alcohol exposure, whereas organisms that increased membrane rigidity were less tolerant. Altered lipid composition was a common response to alcohol exposure, with the most tolerant organisms maintaining a modestly fluid membrane. Our results demonstrate that increased membrane fluidity is not the sole cause of growth inhibition and that alcohols may also denature proteins within the membrane and cytosol, adversely affecting metabolism and decreasing cell growth.     

The relationship between environmental temperature, cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus.

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes. Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might directly be determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.     

Growth of the chorioallantoic membrane into a rapid-prototyped model pore system: experiments and mathematical model

This paper presents a mathematical model to describe the growth of tissue into a rapid-prototyped porous scaffold when it is implanted onto the chorioallantoic membrane (CAM). The scaffold was designed to study the effects of the size and shape of pores on tissue growth into conventional tissue engineering scaffolds, and consists of an array of pores each having a pre-specified shape. The experimental observations revealed that the CAM grows through each pore as an intact layer of tissue, provided the width of the pore exceeds a threshold value. Based on these results a mathematical model is described to simulate the growth of the membrane, assuming that the growth is a function of the local isotropic membrane tension. The model predictions are compared against measurements of the extent of membrane growth through the pores as a function of time for pores with different dimensions.     

MEMBRANE SYNTHESIS IN BACILLUS SUBTILIS : III. The Morphological Localization of the Sites of Membrane Synthesis

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.     

The presence of both growth inhibitory and growth stimulatory factors on membranes prepared from mouse liver

Plasma membrane fractions from mouse livers were examined for the presence of growth regulatory peptides. Both growth inhibitory and growth stimulatory factors were found on these membranes. The growth inhibitory component could be enriched by extractions with both dimethylmaleic anhydride and octylglucoside. The growth stimulatory component could be removed from the membrane by either freeze-thaw, high salt, protease or pyrophosphate treatment, indicating that this factor is an extrinsic membrane protein. The existence of these factors on liver membranes provides an easily obtainable source for the large-scale purification of these molecules and may indicate a possible role in normal tissue growth.     

NADH oxidase of plasma membranes

NADH oxidase is a cyanide-resistant and hormone-responsive oxidase intrinsic to the plasma membrane of both plant and animal cells. The activity has many unique characteristics that distinguish it from other oxidases and oxidoreductases of both organelles and internal membranes and from other oxidoreductases of the plasma membrane. Among these are resistance to inhibition by cyanide, catalase, superoxide dismutase, and phenylchloromer-curibenzoate. Activity is stimulated by hormones and growth factors and inhibited by quinone analogs such as piericidin, the flavin antagonist atebrin, and growth inhibiting gangliosides such as G_M3. In marked contact to the NADH-ferricyanide oxidoreductase of the plasma membrane, the NADH oxidase is activated by lysophospholipids and fatty acids, products of phospholipase A₂ action, in a time-dependent manner suggestive of stabilization of an activated form of the enzyme. The hormone-responsive NADH oxidase of the plasma membrane is not a peroxidase and may function as a terminal oxidase to link transfer of electrons from NADH to oxygen at the plasma membrane. The functional significance of the NADH oxidase of the plasma membrane is unknown but some relationship to growth or growth control is indicated. In both animal and plant plasma membranes, the oxidase is activated by growth factors and hormones to which the cells or tissues of origin have functional hormone or growth factor receptors. In addition, substances that inhibit the oxidase, the associated transmembrane reductase or both, inhibit growth. In transformed cells and tissues, the hormone and growth factor responsiveness of the NADH oxidase is reduced or absent. With human keratinocytes which exhibit an increased sensitivity to the anti-proliferative action of both retinoic acid and calcitriol, the NADH oxidase of the plasma membrane is strongly inhibited by these agents and shows the same increased sensitivity. If transfer of electrons from NADH to oxygen across or within the eukaryotic plasma membrane is an important aspect of growth or growth control, then the hormone- and growth factor-responsive NADH oxidase associated with the plasma membrane could be of fundamental importance. Because of its low basal activity, stimulation by growth factors and hormones, and the inhibition of growth in direct proportion to inhibition of the oxidase, the activity is a candidate as a rate-limiting step in the growth process. Completely unknown is the mechanism whereby NADH oxidization and growth or growth control may be coupled. This, together with further characterization of the activity and the mechanism of loss of control with neoplastic transformation, represent important challenges for future investigations.     

Mechanics and dynamics of actin-driven thin membrane protrusions

Motile cells explore their surrounding milieu by extending thin dynamic protrusions, or filopodia. The growth of filopodia is driven by actin filament bundles that polymerize underneath the cell membrane. We compute the mechanical and dynamical features of the protrusion growth process by explicitly incorporating the flexible plasma membrane. We find that a critical number of filaments are needed to generate net filopodial growth. Without external influences, the filopodium can extend indefinitely up to the buckling length of the F-actin bundle. Dynamical calculations show that the protrusion speed is enhanced by the thermal fluctuations of the membrane; a filament bundle encased in a flexible membrane grows much faster. The protrusion speed depends directly on the number and spatial arrangement of the filaments in the bundle and whether the filaments are tethered to the membrane. Filopodia also attract each other through distortions of the membrane. Spatially close filopodia will merge to form a larger one. Force-velocity relationships mimicking micromanipulation experiments testing our predictions are computed.     

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.     

Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth

The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.     

Glucose modulates basement membrane fibroblast growth factor-2 via alterations in endothelial cell permeability

The effects of glucose extremes on vascular physiology and endothelial cell function have been examined across a range of time scales. Not unexpectedly, chronic glucose exposure induces long term tissue effects. Yet short term exposure can also impose lasting consequences. The persistence of vascular pathology after euglycemic restoration further suggests a glucose exposure memory. Slow turnover reservoirs such as basement membrane are candidates for prolongation of acute events. We hypothesized that glucose-induced vascular dysfunction is related to altered vasoactive compound handling within the endothelial cell-basement membrane co-regulatory unit. Endothelial cell basement membrane-associated fibroblast growth factor-2 increased linearly with culture glucose within days of elevated glucose exposure. Surprisingly, basement membrane fibroblast growth factor-2 binding kinetics remained unchanged. The glucose-induced increase in basement membrane fibroblast growth factor-2 was instead related to enhanced endothelial cell fibroblast growth factor-2 release and permeability. Cellular fibroblast growth factor-2 release occurred concomitant with apoptosis but was not blocked by caspase inhibitors. These data suggest that release was associated with sub-lethal early apoptotic cell membrane damage, perhaps related to reactive oxygen species formation. High glucose basement membrane in turn enhanced endothelial cell proliferation in a fibroblast growth factor-2-dependent manner. We now show that glucose-induced alterations in endothelial cell function promote changes in basement membrane composition, and these changes further affect endothelial cell function. These data highlight the interrelationship of cell and basement membrane in pathological conditions such as hyperglycemia. These phenomena may explain long term effects on the endothelium of short term exposure to glucose extremes.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

The relationship between environmental temperature,cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes.Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might be directly determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Rapid tip growth: insights from pollen tubes

Pollen tubes extend rapidly in an oscillatory manner by the extreme form of polarized growth, tip growth, and provide an exciting system for studying the spatiotemporal control of polarized cell growth. The Rho-family ROP GTPase is a key signaling molecule in this growth control and is periodically activated at the apical plasma membrane to spatially define the apical growth region and temporally precede the burst of growth. The spatiotemporal dynamics of ROP GTPase is interconnected with actin dynamics and polar exocytosis that is required for tip-targeted membrane and wall expansion. Recent advances in the study of the mechanistic interlinks between ROP-centered signaling and spatiotemporal dynamics of cell membrane and wall remodeling will be discussed.