Regulation Of Membrane Dynamics Research Articles

Interaction of microtubules and actin with the N-terminus of βPix-bL directs cellular pinocytosis

βPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, βPix-b(L), was identified as an alternative translational product of the βPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of βPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of βPix-b(L). The results of this study demonstrate that βPix is composed of 24 exons and 21 introns spanning around 100kb. RT-PCR experiments revealed that there are two forms of βPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from βPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of βPix-b(L) revealed that βPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled βPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of βPix-b(L) was also critical for its localization to cellular vesicles. Functionally, βPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of βPix-b(L) in the regulation of membrane dynamics.

Role of annexin A6 isoforms in catecholamine secretion by PC12 cells: Distinct influence on calcium response

Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca(2+)] increases in response to cell excitation and triggers a signal-initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca(2+)-regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock-down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6-1 and AnxA6-2 overexpression reduced Delta[Ca(2+)](c) upon depolarization by 32% and 58%, respectively, while AnxA6 knock-down increased Delta[Ca(2+)](c) by 44%. The mechanism of AnxA6 action on Ca(2+) signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells.

A Vibrio Effector Protein Is an Inositol Phosphatase and Disrupts Host Cell Membrane Integrity

The marine bacterium Vibrio parahaemolyticus causes gastroenteritis in humans and encodes the type III effector protein VPA0450, which contributes to host cell death caused by autophagy, cell rounding, and cell lysis. We found that VPA0450 is an inositol polyphosphate 5-phosphatase that hydrolyzed the D5 phosphate from the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate. VPA0450 disrupted cytoskeletal binding sites on the inner surface of membranes of human cells and caused plasma membrane blebbing, which compromised membrane integrity and probably contributed to cell death by facilitating lysis. Thus, bacterial pathogens can disrupt adaptor protein-binding sites required for proper membrane and cytoskeleton dynamics by altering the homeostasis of membrane-bound inositol-signaling molecules.

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome deficiencies; when its expression is repressed, proteasome deficiencies intensify. Arp2/3 is best known for regulating membrane dynamics and vesicular transport; thus, we performed photobleaching experiments and showed that proteasomes are readily imported into the nucleus but exit the nucleus slowly. Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access substrates throughout the cell, allowing them to effectively regulate cell-compartment-specific activities.

Engineered allosteric activation of kinases in living cells

Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity, but does not affect other protein interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases.

Stress-induced Synthesis of Phosphatidylinositol 3-Phosphate in Mycobacteria

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [(3)H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.

The Hippo tumor-suppressor pathway regulates apical-domain size in parallel to tissue growth

The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.

Cholesterol‐regulated stress fiber formation

Dynamic interactions between cellular membranes and the cytoskeleton are known to play major roles in many cellular responses to environmental cues. External signals resulting in proliferation, differentiation, polarization, and motility must be translated from chemical signals into changes of state, often involving the cytoskeleton-dependent altering of cell shape and redistribution of molecules. Cholesterol, a critical component of eukaryotic cell membranes, performs vital roles in regulating membrane dynamics and function. Here we demonstrate, using mesenchymal and epithelial cell lines, that depletion of membrane cholesterol results in Src kinase-mediated Rho activation and caveolin phosphorylation, which together collaborate to form stress fibers. These results demonstrate that cholesterol is a critical regulator of membrane-cytoskeletal dynamics and suggest that altered cholesterol concentrations may result in dramatic changes in cellular responses mediated by the cytoskeleton.

Force Dependence of a Myo1b Truncation Mutant

Myosin-Is are the single-headed members of the myosin superfamily that associate directly with cell membranes and play roles in regulating membrane dynamics. We previously characterized the force dependence of the widely expressed myosin-I isoform, myo1b, using an optical trap and a novel isometric force clamp. This myo1b isoform, which contains five IQ-motifs, is highly strain sensitive, with forces of 75 fold. We estimated the distance parameter (distance to the transition state of the force dependent step) to be 12 nm by a fit of the force dependence of actin attachment times in the presence of 50 micromolar ATP. In this study, we examine the force dependence of a myo1b truncation mutant that contains only the IQ motif closest to the motor domain. Although the unloaded biochemical kinetics of this mutant are nearly identical to the 5 IQ construct, we found the actin detachment rate to be substantially less force sensitive. These experiments suggest that the length of the regulatory domain modulates force sensitivity.

Annexin A2 Binding to Endosomes and Functions in Endosomal Transport Are Regulated by Tyrosine 23 Phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.

Myosins Under Tension

Myosin I is a single-headed myosin molecule that plays a role in regulating membrane dynamics and structure in eukaryotic cells. Its best-characterized function is to provide tension to sensitize mechanosensitive ion channels responsible for hearing. Myosin I is thought to function by sensing tension and changing its motile properties in response to changes in loads. Laakso et al . used single-molecule measurements to characterize the motor activity of myosin I. Small resisting loads (<2 pN) resulted in a 75 times lower rate of myosin I detachment from actin, dramatically changing its motor properties. This acute sensitivity supports models in which myosin I functions as a molecular force sensor. J. M. Laakso, J. H. Lewis, H. Shuman, E. M. Ostap, Myosin I can act as a molecular force sensor. Science 321 , 133-136 (2008). [Abstract] [Full Text]

Molecular interaction of neurocalcin α with alsin (ALS2)

Membrane microdomains (MDs), or lipid rafts, are recently identified dynamic membrane domains on which various signal-transductions are performed. Intracellular Ca(2+)-binding proteins participate in the Ca(2+) signaling through interaction with various proteins. Neurocalcin alpha (NCalpha) is a member of neuronal calcium sensor (NCS) protein family and shows Ca(2+)-dependent binding to the cell membrane through N-terminal myristoyl moiety. Since NCalpha was identified as a Ca(2+)-dependent binding protein to neuronal MDs, its binding proteins may participate in the signal-transduction on the MDs. In an immunoprecipitate using anti-NCalpha antibody, alsin (ALS2), a protein product of one of the responsive genes for amyotrophic lateral sclerosis, was detected through LC-MS/MS. Specific antibody to alsin was produced and immunoprecipitation using this antibody showed co-sedimentation of NCalpha. Some part of alsin bound to brain-derived MD fraction in the presence of Ca(2+) ions and eluted out by the chelation of Ca(2+) ions, as in the case of NCalpha. Immunostaining of cultured neurons showed broad distribution of alsin and NCalpha, and membrane association of these proteins were increased through Ca(2+) loading by maitotoxin. These results suggest that alsin binds cell membrane in a Ca(2+)-dependent manner through NCalpha and regulates membrane dynamics.

ALS2/alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth

Loss of function mutations in the ALS2 gene account for a number of juvenile/infantile recessive motor neuron diseases, indicating that its gene product, ALS2/alsin, plays a crucial role in maintenance and survival for a subset of neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5 and is implicated in endosome dynamics in cells. However, the role of ALS2 in neurons remains unclear. To elucidate the neuronal ALS2 functions, we investigate cellular phenotypes of ALS2-deficient primary cultured neurons derived from Als2-knockout (KO) mice. Here, we show that ALS2 deficiency results not only in the delay of axon outgrowth in hippocampal neurons, but also in a decreased level of the fluid phase horseradish peroxidase (HRP) uptake, which represents the activity for macropinocytic endocytosis, in cortical neurons. Thus, ALS2 may act as a modulator in neuronal differentiation and/or development through regulation of membrane dynamics.

Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular organisms

Acidification inside membrane compartments is a common feature of all eukaryotic cells. The acidic milieu is involved in many physiological processes including secretion, protein processing, and others. However, its cellular relevance has not been well established beyond the results of in vitro studies involving cultured cell systems. In the last decade, human and mouse genetics have revealed that the acidification machinery is implicated in multiple pathophysiological disorders, and thus our understanding of physiological consequences of the defective acidification in multicellular organisms has improved. In invertebrates including Drosophila and nematodes, mutations of V-ATPase were found to lead the development of rather unexpected phenotypes. Studies have suggested that V-ATPase may be involved in membrane fusion and vesicle formation, important processes for membrane trafficking, and have further implied its involvement in cell-cell fusion. This rather novel idea arose from the phenotypes associated with genetic disorders involving V-ATPase genes in various genetic model systems. In this article, we focus and overview the non-classical, beyond proton-pumping function of the vacuolar-type ATPase in exo/endocytic systems.

Shaping membranes into autophagosomes

The class III phosphatidylinositol 3-kinase (PI(3)KC3) is part of a key complex regulating autophagy. How this complex mechanistically contributes to autophagosome formation remains largely unknown. The BAR-domain-containing protein Bif-1/endophilin B1, commonly associated with the control of membrane curvature, has been identified as a new interacting partner for PI(3)KC3, suggesting the complex plays an active part in the regulation of membrane dynamics during autophagy.

Mechanisms of axon guidance: membrane dynamics and axonal transport in semaphorin signalling

The intricate geometry of neuronal networks poses many unique cell-biological problems regarding the way a growing axon responds to its environment. Several groups of ligand-receptor pairs have been identified to regulate such processes. In this study, we take class 3 semaphorins as an example and review what is known about the intracellular movements of semaphorins throughout neuronal cells, transport support structures and location of release sites. We discuss how their receptor trafficking may contribute to regulate membrane dynamics underlying growth cone motility and the physiological contribution made by class 3 semaphorins-induced acceleration of axoplasmic transport on neurite development.

ARL1 Plays a Role in the Binding of the GRIP Domain of a Peripheral Matrix Protein to the Golgi Apparatus in Plant Cells

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.

REGULATION OF PROTEIN ACTIVITIES BY PHOSPHOINOSITIDE PHOSPHATES

Phosphoinositide phosphates (PIPs) correspond to phosphorylated derivatives of phosphatidylinositol (PI). Despite their relatively low abundance in the plasma membrane, PIPs play a crucial role as precursors of second messengers and are themselves important signaling and targeting molecules. Indeed, modulation of levels of PIPs affects, for example, cortical actin organization, membrane dynamics, and cell migration. The focus of this review is on selected interesting targets of PIPs. Those proteins that bind PIPs and are involved in regulation of actin assembly, actin membrane linkage, and actin contractility are discussed, as well as those that are involved in signaling, such as small GTPases, protein kinases, and phosphatases, or in regulation of membrane dynamics.

Dnm1 forms spirals that are structurally tailored to fit mitochondria

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

Just-right spirals

![][1] Dnm1 self-assembles into spirals tailored to fit mitochondria. Dynamin-related proteins regulate membrane dynamics throughout the cell. Ingerman et al. report on [page 1021][2] that these proteins form large polymers that are structurally tailored to fit their individual jobs