Role In Endocytosis Research Articles

A new role for the dynamin GTPase in the regulation of fusion pore expansion

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.

ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells.

The autosomal recessive hypercholesterolemia protein (ARH) is well known for its role in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs). During uptake, ARH directly binds to the FxNPxY signal in the cytoplasmic tail of LDLR. Interestingly, the same FxNPxY motif is used in basolateral exocytosis of LDLR from recycling endosomes (REs), which is facilitated by the epithelial-specific clathrin adaptor AP-1B. However, AP-1B directly interacts with neither the FxNPxY motif nor the second more distally located YxxØ sorting motif of LDLR. Here, we show that ARH colocalizes and cooperates with AP-1B in REs. Knockdown of ARH in polarized epithelial cells leads to specific apical missorting of truncated LDLR, which encodes only the FxNPxY motif (LDLR-CT27). Moreover, a mutation in ARH designed to disrupt the interaction of ARH with AP-1B specifically abrogates exocytosis of LDLR-CT27. We conclude that in addition to its role in endocytosis, ARH cooperates with AP-1B in basolateral exocytosis of LDLR from REs.

Low-density lipoprotein receptor-related protein 1: new functions for an old molecule

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor member of the low-density lipoprotein (LDL)-receptor family. As LRP1 plays an important role in endocytosis and regulation of signalling pathways, it is implicated in a number of physiologic processes, including the regulation of lipid metabolism, the proliferation of vascular smooth muscle cells and in neuro-development. More recently, LRP1 has been implicated in the catabolism of factor VIII and regulation of its plasma concentrations. The pathophysiology of the role of LRP1 in hemostasis will be summarized in this review.

Secretory Carrier Membrane Protein 2 Regulates Exocytic Insertion of NKCC2 into the Cell Membrane

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.

Proteomic analysis of endocytic vesicles: Rab1a regulates motility of early endocytic vesicles

Texas-Red-asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.

Dimeric Endophilin A2 Stimulates Assembly and GTPase Activity of Dynamin 2

Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high Kd (∼5–15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.

A new Role for the Dynamin Gtpase as a timer that Controls Fusion Pore Expansion

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of the dynamin GTPase activity in controlling fusion pore expansion and post-fusion granule membrane topology was investigated using the powerful membrane curvature-imaging technique of polarized TIRF microscopy and amperometry. A dynamin-1 mutant with increased GTPase activity resulted in faster fusion pore widening and flattening of the granule membrane after exocytosis; dynamin-1 mutants with decreased activity slowed fusion pore widening by stabilizing post-fusion granule membrane curvature. The experiments indicate that the GTPase activity of dynamin functions as a timer determining the rapidity of fusion pore expansion from 10's of milliseconds to seconds after fusion, in addition to its role in endocytosis. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate post-fusion events in exocytosis that control the rate of release of soluble granule contents.

The Role of Monoubiquitination in Endocytic Degradation of Human Ether-a-go-go-related Gene (hERG) Channels under Low K+ Conditions

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.

Purification of Actin from Fission Yeast Schizosaccharomyces pombe and Characterization of Functional Differences from Muscle Actin

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.

Role of Vacuolar ATPase in the Trafficking of Renal Type IIa Sodium-phosphate Cotransporter

Background/Aims: Total body phosphate homeostasis is regulated by expression of type IIa sodium phosphate cotransporter (NpT2a) in the apical membrane (BBM) of renal proximal tubule cells. NpT2a expression is regulated by dietary phosphate and PTH but the mechanisms for trafficking of the protein are unknown. Based on 2D gel electrophoresis and mass spectroscopy data that changes in dietary phosphate stimulated changes in BBM expression of vacuolar H⁺-ATPase, we hypothesized that vacuolar H⁺-ATPase plays a significant role in regulation of NpT2a in opossum kidney (OK) cells, a model for renal proximal tubule transport. Methods: Role of vacuolar H⁺-ATPase was studied in opossum kidney (OK) cells by examining the effect of inhibition of vacuolar H⁺-ATPase on Pi uptake and NpT2a expression. Results: Pretreatment of OK cells with bafilomycin A₁ and concanamycin A, inhibitors of vacuolar H⁺-ATPases, blocked high phosphate- and PTH-induced degradation of NpT2a, but had no effect on high phosphate or PTH induced inhibition of sodium-dependent phosphate transport. Exposure of the cells to bafilomycin A₁ significantly decreased phosphate transport and apical membrane expression of NpT2a. Treatment with brefeldin A, an inhibitor of Golgi transport, decreased phosphate transport and apical membrane expression of NpT2a while treatment of cells with both brefeldin A and bafilomycin A₁ had no additive effect. Conclusion: We conclude that vacuolar H⁺-ATPase plays a significant role in exocytosis of NpT2a into the apical membrane and in degradation of NpT2a but has no role in endocytosis.

Expression of Vps1 I649K, a self-assembly defective yeast dynamin, leads to formation of extended endocytic invaginations

The dynamin proteins have been associated with the process of endocytosis for many years. Until recently it was considered that yeast dynamin-related proteins did not play a role in endocytosis and the proposed scission function of dynamin was attributed to another group of proteins, the amphiphysins. However, it has now been shown that the yeast dynamin-like protein Vps1 shows a transient burst of localization to sites of endocytosis. Vps1 assembles at cortical sites at the time when actin polymerization is proposed to drive plasma membrane invagination. In concert with the amphiphysins Vps1 is then thought to function in the scission step to release a formed vesicle. It was shown that a mutation preventing self assembly of Vps1 caused a defect in endocytosis but not in other functions with which Vps1 is associated. Using electron microscopy we now show that this mutation I649K, corresponding to I690K in human Dyn1, causes formation of long endocytic invaginations. The data suggest that an ability of Vps1 to self assemble and to thereby stimulate its GTPase activity is critical for the 'pinching-off' stage of endocytosis to form a vesicle.

Going on a (Membrane) Bender to the F-BAR

This study is a personal favorite because it simplified the field and proposed an elegant model of how actin and membranes work together in endocytic trafficking. A group of proteins containing FCH domains (Fes/CIP4 homology) were discovered to have extended homology with the banana-shaped alpha helical BAR domain and renamed F-BAR proteins. F-BAR and BAR proteins bind to and bend curved membrane surfaces and participate in budding and tubulation events. Dynamin, a large GTPase that forms helical cages around tubulated membranes, is the main partner of BAR and F-BAR proteins. The actin cytoskeleton opposes scission of the tubulated membranes pending arrival of an appropriate signal. The idea that dynamin is the most important partner of F-BARs and that the trio of actin, F-BAR, and dynamin sculpt and cut membrane tubules in this elegant way remains important for future research and puts into context the barrage of sometimes disparate studies on these proteins. This PaperPick refers to and the Actin Cytoskeleton Cooperatively Regulate Plasma Membrane Invagination by BAR and F-BAR Proteins by T. Itoh, K.S. Erdmann, A. Roux, B. Habermann, H. Werner, and P. De Camilli, published in December, 2005. F-BAR Proteins: Coordinating the Effects of Actin and Dynamin on Endocytosis Pietro De Camilli discusses how his group came to define the F-BAR domain, its role in endocytosis, and where subsequent work has taken the field.

Direct dynamin–actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

Candida albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5

Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida albicans via the Wiskott-Aldrich syndrome gene homologue WAL1. To get a more detailed mechanistic understanding of this link we have investigated a potentially conserved interaction between Wal1 and the C. albicans WASP-interacting protein (WIP) homologue encoded by VRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under serum inducing conditions but filamentation can be observed on Spider medium. Mutant vrp1 cells show a delay in endocytosis - measured as the uptake and delivery of the lipophilic dye FM4-64 into small endocytic vesicles - compared to the wild-type. Vacuolar morphology was found to be fragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1 daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration of the wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we could demonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1, which contains the WH1 domain. Furthermore, we found that Myo5 has several potential interaction sites on Vrp1. This suggests that a Wal1-Vrp1-Myo5 complex plays an important role in endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarized hyphal growth in C. albicans.

Human integrin α(3)β(1) regulates TLR2 recognition of lipopeptides from endosomal compartments.

BackgroundToll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered.Methodology/Principal FindingsUsing shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam3CSK4, are dependent upon an integrin, α3β1. The mechanism for integrin α3β1 involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam3CSK4 is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane.Conclusion/SignificanceHere we identify integrin α3β1 as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α3β1-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α3β1 serves as a mechanism for modulating inflammatory responses.

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.

The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Abstract 3952: CALM deficiency in fit1 embryonic cells alters expression and rate of endocytosis of growth factor receptors

Abstract Background: Gene rearrangements involving the Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and Mixed-Lineage Leukemia (MLL) or AF10 genes have been identified in aggressive leukemias and lymphomas. Expression of CALM-containing fusion proteins immortalizes murine hematopoietic cells in vitro, correlating with leukemogenesis in vivo. Disruption of normal CALM, MLL or AF10 protein function as a result of these translocations likely contributes to transformation, although the precise mechanisms are unknown. The native CALM protein is involved in clathrin-mediated endocytosis (CME); it localizes to the cytoplasmic side of endocytic vesicles, interacts with both membrane elements and clathrin, and when over- or under-expressed, disrupts endocytosis. To better understand the effects of altered CALM activity, we have focused our studies on mutant fit1 mice that lack CALM expression. Previous studies have shown that these mice have iron deficiency, defective hematopoiesis and ultimately premature death. Here we examine CALM's role in endocytosis and cellular function using cells derived from fit1 mice. Objective: To examine the effect of CALM deficiency on cell surface receptor expression and endocytosis using mouse embryonic fibroblasts (MEFs) and fetal liver cells derived from normal and CALM-deficient mice. Design: Day 14 MEFs were generated from normal, heterozygous, and mutant fit1 embryos. Immortalized MEFs were compared in terms of quantity of cell surface and total cellular receptors (by flow cytometry and Western blotting), receptor mRNA levels, and rate of endocytosis. Receptors were also measured in fetal liver derived from the same embryos. Results: When compared to their normal counterparts, cells lacking full length CALM showed altered cell surface receptor expression: transferrin receptor (TfR) protein levels measured by flow cytometry were increased two-fold, while EGF receptor (EGFR) levels were reduced two-fold. These results correlated with increased total cellular TfR protein and mRNA levels and decreased total EGFR protein levels. Whereas the rate of internalization of TfR was similar, the rate of endocytosis of EGFR was significantly reduced in CALM-deficient compared with wildtype cells. Elevated TfR levels were also identified in CALM-deficient fetal liver cells. Conclusions: CALM deficiency in fit1-derived cells results in increased TfR surface expression; this is consistent with the iron deficiency phenotype of fit1 mice. The reduced surface expression of EGFR seen in fit1 cells may be a compensatory mechanism that counteracts the increased growth factor receptor signaling that is associated with impaired endocytosis. Since CALM haploinsufficiency is a feature of CALM-AF10 and MLL-CALM leukemias, our results suggest that the perturbation of normal growth factor biology may contribute to transformation in these malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3952.

Abstract 1062: Numb, an intrinsic Notch inhibitor, possesses a tumor suppressor role through regulation of Plk1 localization during mitosis

Abstract Numb, an intrinsic Notch inhibitor and cell fate determinant, has been shown to possess many functions in proper cell division. These include its role in endocytosis, a tumor suppressor role via regulation of p53 stability, and asymmetric self renewal during progenitor cell division. Studies have shown that a proper localization of fully functional Plk1 is required for mitotic entry, progression and exit. Plk1 has been shown to play a critical role in bi-polar spindle formation and maturation during mitosis. It has been shown that chemical inhibition as well as genetic knockdown of Plk1 results in cells forming a mono-polar spindle phenotype surrounded by DNA rather than a bi-polar arrangement on either side of the metaphase plate. This severe mitotic phenotype results in a G2/M cell cycle arrest and mitotic catastrophe. Also, milder inhibition of Plk1 has been shown to result in errors in bi-polar spindle formation including multi-polar phenotypes or incomplete microtubule attachment and division. Some of these less pronounced phenotypes may not prevent cell division and may contribute to cellular aneuploidy and cancer. In this study, employing melanoma cell lines A375 and HS294T, we demonstrated that Numb and Plk1 interact as shown by co-immunoprecipitation and immunofluorescent assays. In addition, we found that both Plk1 and Numb co-localize to the spindle poles in metaphase. Further, using lentiviral shRNA knockdown of Plk1 or Numb, we also found that loss of Plk1 or Numb dysregulates the localization of the other. Most striking was the pronounced mislocalization of Plk1 from the spindle poles. Although, the majority of metaphase cells were still bi-polar, there was a marked increase in mislocalized γ-tubulin in some instances. Additionally, in the cells with two defined spindle poles, γ-tubulin still appears to be less pronounced at strictly the spindle pole with an apparent indistinct staining surrounding the poles indicating that Numb may not be directly required for γ-tubulin localization to the spindles but the misregulated Plk1 localization slows or prevents spindle pole maturation and γ-tubulin recruitment. Conversely, the differing phenotypes of γ-tubulin localization may also be explained by a redundant role of Numb-like, though this is yet to be explored. Overall, these data suggested that Numb may possess tumor suppressor functions by regulating proper Plk1 localization and proper spindle pole maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1062.