Large GTPase Dynamin Research Articles

Dynamin-2 mutations associated with centronuclear myopathy are hypermorphic and lead to T-tubule fragmentation.

Skeletal muscle requires adequate membrane trafficking and remodeling to maintain its normal structure and functions. Consequently, many human myopathies are caused by mutations in membrane trafficking machinery. The large GTPase dynamin-2 (Dyn2) is best known for catalyzing membrane fission during clathrin-mediated endocytosis (CME), which is critical for cell signaling and survival. Despite its ubiquitous expression, mutations of Dyn2 are associated with two tissue-specific congenital disorders: centronuclear myopathy (CNM) and Charcot-Marie-Tooth (CMT) neuropathy. Several disease models for CNM-Dyn2 have been established to study its pathogenic mechanism; yet the cellular and biochemical effects of these mutations are still not fully understood. Here we comprehensively compared the biochemical activities of disease-associated Dyn2 mutations and found that CNM-Dyn2 mutants are hypermorphic with enhanced membrane fission activity, whereas CMT-Dyn2 is hypomorphic. More importantly, we found that the expression of CNM-Dyn2 mutants does not impair CME in myoblast, but leads to T-tubule fragmentation in both C2C12-derived myotubes and Drosophila body wall muscle. Our results demonstrate that CNM-Dyn2 mutants are gain-of-function mutations, and their primary effect in muscle is T-tubule disorganization, which explains the susceptibility of muscle to Dyn2 hyperactivity.

Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

Overexpression of N-terminal kinase like gene promotes tumorigenicity of hepatocellular carcinoma by regulating cell cycle progression and cell motility.

Amplification and overexpression of CHD1L is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC). Here we found that one of CHD1L downstream targets, NTKL, was frequently upregulated in HCC, which was significantly correlated with vascular invasion (P = 0.012) and poor prognosis (P = 0.050) of HCC. ChIP assay demonstrated the binding of CHD1L to the promoter region of NTKL. QRT-PCR study showed that the expression of NTKL positively correlated with CHD1L expression in both clinical samples and cell lines. Functional study found that NTKL had strong oncogenic roles, including increased cell growth, colony formation in soft agar, and tumor formation in nude mice. Further study found that NTKL could promote G1/S transition by decreasing P53 and increasing CyclinD1 expressions. NTKL overexpression could accelerate the mitotic exit and chromosome segregation, which led to the cytokinesis failure and subsequently induced apoptosis. NTKL also regulated cell motility by facilitating philopodia and lamellipodia formation through regulating F-actin reorganization and the phosphorylation of small GTPase Rac1/cdc42. Using co-IP and mass spectrometry approach, we identified the large GTPase dynamin2 as an interacting protein of NTKL, which might be responsible for the phenotype alterations caused by NTKL overexpression, such as cytokinesis failure, increased cell motility and abnormal of cell division.

Role of dynamin in elongated cell migration in a 3D matrix

The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

Dynamin2 organizes lamellipodial actin networks to orchestrate lamellar actomyosin.

Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.

Dynamin-dependent maintenance of epithelial integrity is essential for zebrafish epiboly

Epiboly, the thinning and spreading of one tissue over another, is a widely employed morphogenetic movement that is essential for the development of many organisms. In the zebrafish embryo, epiboly describes the coordinated vegetal movement of the deep cells, enveloping layer (EVL) and yolk syncytial layer (YSL) to engulf the yolk cell. Recently, we showed that the large GTPase Dynamin plays a fundamental role in epiboly in the early zebrafish embryo. Because Dynamin plays a well-described role in vesicle scission during endocytosis, we predicted that Dynamin might regulate epiboly through participating in bulk removal of the yolk cell membrane ahead of the advancing margin, a proposed part of the epiboly motor. Unexpectedly, we found that Dynamin function was dispensable in the yolk cell and instead, it was required to maintain the epithelial integrity of the EVL during epiboly. Here, we present a model describing the maintenance of EVL integrity, which is required for the proper generation and transmission of tension during epiboly. Furthermore, we discuss the role of Dynamin-mediated regulation of ezrin-radixin-moesin (ERM) family proteins in the maintenance of epithelial integrity.

Essential Function of Dynamin in the Invasive Properties and Actin Architecture of v-Src Induced Podosomes/Invadosomes

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.

Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is poorly characterized. We hypothesized that the large GTPase dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment.

Muscle-specific function of the centronuclear myopathy and Charcot–Marie–Tooth neuropathy-associated dynamin 2 is required for proper lipid metabolism, mitochondria, muscle fibers, neuromuscular junctions and peripheral nerves

The ubiquitously expressed large GTPase Dynamin 2 (DNM2) plays a critical role in the regulation of intracellular membrane trafficking through its crucial function in membrane fission, particularly in endocytosis. Autosomal-dominant mutations in DNM2 cause tissue-specific human disorders. Different sets of DNM2 mutations are linked to dominant intermediate Charcot-Marie-Tooth neuropathy type B, a motor and sensory neuropathy affecting primarily peripheral nerves, or autosomal-dominant centronuclear myopathy (CNM) presenting with primary damage in skeletal muscles. To understand the underlying disease mechanisms, it is imperative to determine to which degree the primary affected cell types require DNM2. Thus, we used cell type-specific gene ablation to examine the consequences of DNM2 loss in skeletal muscle cells, the major relevant cell type involved in CNM. We found that DNM2 function in skeletal muscle is required for proper mouse development. Skeletal muscle-specific loss of DNM2 causes a reduction in muscle mass and in the numbers of muscle fibers, altered muscle fiber size distributions, irregular neuromuscular junctions (NMJs) and isolated degenerating intramuscular peripheral nerve fibers. Intriguingly, a lack of muscle-expressed DNM2 triggers an increase of lipid droplets (LDs) and mitochondrial defects. We conclude that loss of DNM2 function in skeletal muscles initiates a chain of harmful parallel and serial events, involving dysregulation of LDs and mitochondrial defects within altered muscle fibers, defective NMJs and peripheral nerve degeneration. These findings provide the essential basis for further studies on DNM2 function and malfunction in skeletal muscles in health and disease, potentially including metabolic diseases such as diabetes.

Dynamin 2 Potentiates Invasive Migration of Pancreatic Tumor Cells through Stabilization of the Rac1 GEF Vav1

The large GTPase Dynamin 2 (Dyn2) is markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. Here we demonstrate an unexpected mechanism of Dyn2 action in these contexts via direct binding to the Rac1 guanine nucleotide exchange factor (GEF) Vav1. Surprisingly, disruption of the Dyn2-Vav1 interaction targets Vav1 to the lysosome for degradation via an interaction with the cytoplasmic chaperone Hsc70, resulting in a dramatic reduction of Vav1 protein stability. Importantly, a specific mutation in Vav1 near its Dyn2-binding C-terminal Src homology 3 (SH3) domain prevents Hsc70 binding, resulting in a stabilization of Vav1 levels. Dyn2 binding regulates the interaction of Vav1 with Hsc70 to control the stability and subsequent activity of this oncogenic GEF. These findings elucidate how Dyn2 activates Rac1, lamellipod protrusion, and invasive cellular migration and provide insight into how this specific Vav is ectopically expressed in pancreatic tumors.

A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis

AbstractAbstract 608Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia. Disclosures:No relevant conflicts of interest to declare.

407a Oncogenic AKT1 Regulation by Tumor Suppressor KLF11: Implications in Growth Regulation During Neoplastic Transformation in Barrett's Esophagus and Interference by Carcinogenic Bile Acid Dependent Erk2 and PKCa Activation

Pancreatic adenocarcinoma is the fifth leading cause of cancer death, with an abysmal 5year survival rate of less than 5%, due in large part to the high incidence of metastasis. Metastatic invasion represents a complex synergy of cell motility, adhesion, and matrix remodeling. How these multiple processes are coordinated during dissemination is a central focus of tumor cell biology. The small oncogenic G protein Rac1 is a major regulator of cell motility, and it is activated through the action of guanine nucleotide exchange factors (GEFs). A critical GEF in tumor cell biology is the proto-oncogene Vav1, which is ectopically expressed in most pancreatic tumors to cause poor prognosis in patients and enhanced tumor growth In Vitro. Vav1 interacts directly with the large GTPase Dynamin 2 (Dyn2), though the function of this interaction is not known. Importantly, Dyn2 is also markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. We have recently found that Dyn2 binds Vav1 directly in pancreatic tumor cells. Therefore, the GOAL of this study was to test if the Dyn2Vav1 interaction could promote the activation of Rac1 and the invasive migration of pancreatic tumor cells. RESULTS: We found that disruption of the Dyn2-Vav1 interaction impairs Rac1 activation and subsequent lamellipodia formation and cell migration, indicating that Dyn2-Vav1 binding regulates tumor cell motility. Surprisingly, RNAi-mediated suppression of Dyn2 or disruption of Dyn2-Vav1 binding results in a dramatic reduction in Vav1 protein stability, resulting in diminished levels of this proto-oncogene and decreased Rac1 activation and migration. Remarkably, this degradative reduction is a result of Vav1 targeted transport to the lysosome, as treatment of these cells with the protease inhibitor chloroquine prevents Vav1 loss, and immunofluorescent cell staining shows a dramatic localization of Vav1 to the lysosomal lumen when it cannot bind Dyn2.CONCLUSION: These findings demonstrate a novel regulatory role for direct Dyn2 binding in Rac1 activation, lamellipod protrusion and cellular migration through the stabilization of Vav1, and demonstrate a potent mechanism by which metastatic migration is upregulated in pancreatic tumor cells. This study was funded by NCI CA104125 to MAM.

407 Rock-Inhibitors Reduce the Induction of Epithelial to Mesenchymal Transition, Migration and Invasion Caused by Bone Morphogentic Protein Signaling in SMAD4 Deficient Colorectal Cancers

Pancreatic adenocarcinoma is the fifth leading cause of cancer death, with an abysmal 5year survival rate of less than 5%, due in large part to the high incidence of metastasis. Metastatic invasion represents a complex synergy of cell motility, adhesion, and matrix remodeling. How these multiple processes are coordinated during dissemination is a central focus of tumor cell biology. The small oncogenic G protein Rac1 is a major regulator of cell motility, and it is activated through the action of guanine nucleotide exchange factors (GEFs). A critical GEF in tumor cell biology is the proto-oncogene Vav1, which is ectopically expressed in most pancreatic tumors to cause poor prognosis in patients and enhanced tumor growth In Vitro. Vav1 interacts directly with the large GTPase Dynamin 2 (Dyn2), though the function of this interaction is not known. Importantly, Dyn2 is also markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. We have recently found that Dyn2 binds Vav1 directly in pancreatic tumor cells. Therefore, the GOAL of this study was to test if the Dyn2Vav1 interaction could promote the activation of Rac1 and the invasive migration of pancreatic tumor cells. RESULTS: We found that disruption of the Dyn2-Vav1 interaction impairs Rac1 activation and subsequent lamellipodia formation and cell migration, indicating that Dyn2-Vav1 binding regulates tumor cell motility. Surprisingly, RNAi-mediated suppression of Dyn2 or disruption of Dyn2-Vav1 binding results in a dramatic reduction in Vav1 protein stability, resulting in diminished levels of this proto-oncogene and decreased Rac1 activation and migration. Remarkably, this degradative reduction is a result of Vav1 targeted transport to the lysosome, as treatment of these cells with the protease inhibitor chloroquine prevents Vav1 loss, and immunofluorescent cell staining shows a dramatic localization of Vav1 to the lysosomal lumen when it cannot bind Dyn2.CONCLUSION: These findings demonstrate a novel regulatory role for direct Dyn2 binding in Rac1 activation, lamellipod protrusion and cellular migration through the stabilization of Vav1, and demonstrate a potent mechanism by which metastatic migration is upregulated in pancreatic tumor cells. This study was funded by NCI CA104125 to MAM.

406 Invasive Migration of Pancreatic Tumor Cells is Potentiated by a Novel Stabilization of the Oncogenic RAC GEF Vav1 by the Large GTPase Dynamin 2

406 Invasive Migration of Pancreatic Tumor Cells is Potentiated by a Novel Stabilization of the Oncogenic RAC GEF Vav1 by the Large GTPase Dynamin 2

Mutation spectrum in the large GTPase dynamin 2, and genotype-phenotype correlation in autosomal dominant centronuclear myopathy

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT.

Membrane Fission: The Biogenesis of Transport Carriers

Membrane-bound transport carriers are used to transfer cargo between membranes of the secretory and the endocytic pathways. The generation of these carriers can be classified into three steps: segregation of cargo away from the residents of a donor compartment (cargo sorting), generation of membrane curvature commensurate with the size of the cargo (membrane budding or tubulation), and finally separation of the nascent carrier from the donor membrane by a scission or membrane fission event. This review summarizes advances in our understanding of some of the best-characterized proteins required for the membrane fission that separates a transport carrier from its progenitor compartment: the large GTPase dynamin, the small guanine nucleotide-binding (G) proteins of the Arf family, BAR (Bin-amphiphysin-Rvs) domain proteins, and protein kinase D. These proteins share their ability to insert into membranes and oligomerize to create the large curvatures; however, the overall process of fission that involves these proteins appears to be quite different.

Arp2/3 mediates early endosome dynamics necessary for the maintenance of PAR asymmetry in Caenorhabditis elegans.

The widely conserved Arp2/3 complex regulates branched actin dynamics that are necessary for a variety of cellular processes. In Caenorhabditis elegans, the actin cytoskeleton has been extensively characterized in its role in establishing PAR asymmetry; however, the contributions of actin to the maintenance of polarity before the onset of mitosis are less clear. Endocytic recycling has emerged as a key mechanism in the dynamic stabilization of cellular polarity, and the large GTPase dynamin participates in the stabilization of cortical polarity during maintenance phase via endocytosis in C. elegans. Here we show that disruption of Arp2/3 function affects the formation and localization of short cortical actin filaments and foci, endocytic regulators, and polarity proteins during maintenance phase. We detect actin associated with events similar to early endosomal fission, movement of endosomes into the cytoplasm, and endosomal movement from the cytoplasm to the plasma membrane, suggesting the involvement of actin in regulating processes at the early endosome. We also observe aberrant accumulations of PAR-6 cytoplasmic puncta near the centrosome along with early endosomes. We propose a model in which Arp2/3 affects the efficiency of rapid endocytic recycling of polarity cues that ultimately contributes to their stable maintenance.

Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

Author SummaryDuring animal development and in adulthood many cells are programmed to die by an active process called apoptosis. These dead or dying apoptotic cells are swiftly taken up by scavenger cells into membrane-bound compartments—phagosomes—where they are subsequently degraded when other intracellular organelles containing digestive enzymes fuse with phagosomes—a process called phagosome maturation. Phagocytosis of apoptotic cells is important for tissue remodeling in development and to prevent harmful inflammatory and autoimmune responses. In nematode worms—a model organism in which to study apoptosis—phagosome maturation is accompanied by two waves of the signaling molecule phosphatidylinositol 3-phosphate (PtdIns(3)P) in this compartment: one that forms soon after the formation of the phagosome and lasts for 10–15 minutes, and a second, weaker one 10 minutes later that lasts until the apoptotic cell is fully digested. In this study, we investigated the mechanism that regulates the timing and length of these two waves. We found that they are established by the sequential and combined action of three enzymes: two phosphoinositide 3-kinases, which add a phosphate group to the 3′ site of PtdIns, and one phosphoinositide 3-phosphatase, which removes it. We showed that inactivation of both kinases depleted phagosomes of PtdIns(3)P and resulted in the arrest of phagosome maturation and degradation of apoptotic cells. In addition, the timely turnover of PtdIns(3)P catalyzed by the phosphatase was critical for the step-wise progress of phagosome maturation. Our findings suggest that reversible phosphorylation of phophoinositides, catalyzed by distinct sets of kinases and phosphatases, might be a general mechanism to drive multi-step intracellular membrane trafficking events.

Chapter eight - Membrane Trafficking and Phagosome Maturation During the Clearance of Apoptotic Cells

Apoptosis is a cellular suicide process that quietly and efficiently eliminates unwanted or damaged cells. In metazoans, cells that undergo apoptosis are swiftly internalized by phagocytes and subsequently degraded inside phagosomes through phagosome maturation, a process that involves the fusion between phagosomes and multiple kinds of intracellular organelles and the gradual acidification of phagosomal lumen. In recent years, rapid progress has been made, in particular, through studies conducted in the model organism, the nematode Caenorhabditis elegans, in understanding the membrane trafficking events and molecular mechanisms that govern the degradation of apoptotic cells through phagosome maturation. These studies revealed the novel and essential functions of a large number of proteins, including the large GTPase dynamin, multiple Rab small GTPases and their regulatory proteins, the lipid second messenger PtdIns(3)P and its effectors, and unexpectedly, the phagosomal receptors for apoptotic cells, in promoting phagosome maturation. Further, novel signaling pathways essential for phagosome maturation have been delineated. Here, we discuss these exciting new findings, which have significantly deepened and broadened our understanding of the mechanisms that regulate the interaction between intracellular organelles and phagosomes.

Disruption of Microtubule Network Rescues Aberrant Actin Comets in Dynamin2-Depleted Cells

A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.