Search

Phagocytosis of invading microorganisms by professional phagocytic cells has a central role in innate immunity. However, several microorganisms developed strategies to subvert this process. Previously, we reported that bacteria and protozoa modulate differently the expression of Rab GTPases. Moreover, our results suggested that this modulation can contribute to avoid phagocytosis. Here, we investigated the mechanism by which the malaria parasite Plasmodium berghei and the bacterium Escherichia coli subvert phagocytosis through the modulation of Rab14 or Rab9a expression, respectively. We first confirmed that the scavenger receptor CD36 and the Toll-like receptor (TLR) 4 are required for the phagocytosis of P. berghei and E. coli, respectively. Interestingly, we observed that Rab14 silencing leads to an increase in the surface expression of CD36 in macrophages, which can explain the increase in the phagocytosis of P. berghei we reported previously. Similar results were obtained for Rab9a and TLR4, i.e. Rab9a silencing causes an upregulation of TLR4 surface expression in macrophages. Furthermore, we found that the decrease in the internalization of CD36 and TLR4, upon Rab14 or Rab9a silencing, respectively, can explain the increase in the surface levels of these receptors. Thus, our studies provide evidence that the modulation of phagocytosis caused by changes in Rab expression is operated, at least partly through changes in the surface levels of phagocytic receptors.

Extracellular vesicles, including exosomes, have recently been implicated as novel mediators of immune cell communication in mammals. However, roles for endogenously produced exosomes in regulating immune cell functions in vivo are just beginning to be identified. In this article, we demonstrate that Rab27a and Rab27b double-knockout (Rab27DKO) mice that are deficient in exosome secretion have a chronic, low-grade inflammatory phenotype characterized by elevated inflammatory cytokines and myeloproliferation. Upon further investigation, we found that some of these phenotypes could be complemented by wild-type (WT) hematopoietic cells or administration of exosomes produced by GM-CSF-expanded bone marrow cells. In addition, chronically inflamed Rab27DKO mice had a blunted response to bacterial LPS, resembling endotoxin tolerance. This defect was rescued by bone marrow exosomes from WT, but not miR-155 -/- , cells, suggesting that uptake of miR-155-containing exosomes is important for a proper LPS response. Further, we found that SHIP1 and IRAK-M, direct targets of miR-155 that are known negative regulators of the LPS response, were elevated in Rab27DKO mice and decreased after treatment with WT, but not miR-155 -/- , exosomes. Together, our study finds that Rab27-dependent exosome production contributes to homeostasis within the hematopoietic system and appropriate responsiveness to inflammatory stimuli., (Copyright © 2017 by The American Association of Immunologists, Inc.)

In a variety of cells, secretory processes require the activation of both Rab27a and L-type channels of the Ca(V)1.3 subtype. In the retinal pigment epithelium (RPE), Rab27a and Ca(V)1.3 channels regulate growth-factor secretion towards its basolateral side. Analysis of murine retina sections revealed a co-localization of both Rab27a and Ca(V)1.3 at the basolateral membrane of the RPE. Heterologously expressed Ca(V)1.3/β3/α2δ1 channels showed negatively shifted voltage-dependence and decreased current density of about 70% when co-expressed with Rab27a. However, co-localization analysis using α(5)β(1) integrin as a membrane marker revealed that Rab27a co-expression reduced the surface expression of Ca(V)1.3 only about 10%. Physical binding of heterologously expressed Rab27a with Ca(V)1.3 channels was shown by co-localization in immunocytochemistry as well as co-immunoprecipitation which was abolished after deletion of a MyRIP-homologous amino acid sequence at the II-III linker of the Ca(V)1.3 subunit. Rab27a over-expression in ARPE-19 cells positively shifted the voltage dependence, decreased current density of endogenous Ca(V)1.3 channels and reduced VEGF-A secretion. We show the first evidence of a direct functional modulation of an ion channel by Rab27a suggesting a new mechanism of Rab and ion channel interaction in the control of VEGF-A secretion in the RPE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Neutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated., Results: Here we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model., Conclusions: These data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.

The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a(+) structures, some of which colocalized with CD63(+) crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.

Rab and ADP-ribosylation factor (Arf) family proteins are master regulators of membrane trafficking and are involved in all steps of vesicular transport. These families of small guanine-nucleotide-binding (G) proteins are well suited to regulate membrane trafficking processes since their nucleotide state determines their conformation and the capacity to bind to a multitude of effectors, which mediate their functions. In recent years, several inherited diseases have been associated with mutations in genes encoding proteins belonging to these two families or in proteins that regulate their GTP-binding cycle. The genetic diseases that are caused by defects in Rabs, Arfs or their regulatory proteins are heterogeneous and display diverse symptoms. However, these diseases mainly affect two types of subcellular compartments, namely lysosome-related organelles and cilia. Also, several of these diseases affect the nervous system. Thus, the study of these diseases represents an opportunity to understand their etiology and the molecular mechanisms involved, as well as to develop novel therapeutic strategies., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, Rab27a primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting Rab27a-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and Rab27a/b double knockout BMMCs highlighted a secondary positive role for Rab27a in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosin Va (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double Rab27a/b knockout secretory impairment. We conclude that Rab27a and Rab27b regulate distinct steps in the BMMC degranulation pathway, with Rab27a/Mlph/MyoVa regulating cortical actin stability upstream of Rab27a/b/Munc13-4-dependent granule exocytosis., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.

Rab GTPases are master regulators of intracellular trafficking and, in recent years, their role in the control of different aspects of tumour progression has emerged. In the present review, we show that Rab GTPases are disregulated in many cancers and have central roles in tumour cell migration, invasion, proliferation, communication with stromal cells and the development of drug resistance. As a consequence, Rab proteins may be novel potential candidates for the development of anticancer drugs and, in this context, the preliminary results obtained with an inhibitor of Rab function are also discussed.

During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context., (©2012 AACR.)

Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

Regulated secretory pathways coordinated by small Rab GTPases are critically involved in intercellular communications. Here, we report the expression and localization of Rab27B in developing and differentiated epithelial human tissues by immunohistochemistry. Rab27B is poorly expressed in fetal tissues suggesting that several developmental mechanisms involved in epithelial differentiation and functions are mediated by other secretory Rab GTPases, such as Rab27A or Rab3 family members. In adult tissues, Rab27B is expressed in a wide variety of differentiated secretory epithelial cells, including those lining the salivary gland, gastrointestinal, mammary and prostate tracts. The complex pattern of Rab27B expression indicates that dysregulation of Rab27B-mediated secretion may have profound implications for disease pathology.

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport., (© 2011 John Wiley & Sons A/S.)

Von-Willebrand factor (vWF) is a highly multimerized hemostatic glycoprotein that is stored in endothelial Weibel-Palade bodies (WPB) and secreted upon cell stimulation to act in recruiting platelets to sites of vessel injury. Only fully matured multimeric vWF represents an efficient anchor for platelets, and endothelial cells have developed mechanisms to prevent release of immature vWF. Full maturation of vWF occurs within WPB following their translocation from a perinuclear site of emergence at the trans-Golgi network (TGN) to the cell periphery. The WPB-associated small GTPase Rab27a is involved in restricting immature WPB exocytosis and we searched for links between Rab27a and the actin cytoskeleton that could anchor WPB inside endothelial cells until they are fully matured. We here identify myosin Va as such link. Myosin Va forms a tripartite complex with Rab27a and its effector MyRIP and depletion of or dominant-negative interference with myosin Va leads to an increase in the ratio of perinuclear to more peripheral WPB. Concomitantly, myosin Va depletion results in an elevated secretion of less-oligomeric vWF from histamine-stimulated endothelial cells. These results indicate that a Rab27a/MyRIP/myosin Va complex is involved in linking WPB to the peripheral actin cytoskeleton of endothelial cells to allow full maturation and prevent premature secretion of vWF., (© 2011 John Wiley & Sons A/S.)

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins., (© 2011 John Wiley & Sons A/S.)

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.

In contemporary oncology practice, an urgent need remains to refine the prognostic assessment of breast cancer. It is still difficult to identify patients with early breast cancer who are likely to benefit from adjuvant chemotherapy. Although invasion of cancer cells is the main prognostic denominator in tumor malignancy, our molecular understanding and diagnosis are often inadequate to cope with this activity. Therefore, deciphering molecular pathways of how tumors invade and metastasize may help in the identification of a useful prognostic marker. We recently discovered that the secretory small GTPase Rab27B, a regulator of vesicle exocytosis, delivers proinvasive signals for increased invasiveness, tumor size, and metastasis of various estrogen receptor (ER)-positive breast cancer cell lines, both in vitro and in vivo. In human breast cancer specimens, the presence of Rab27B protein proved to be associated with a low degree of differentiation and the presence of lymph node metastasis in ER-positive breast cancer.

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a 'prenylation block-and-release' assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Background: Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood., Results: We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7., Conclusions: Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.

Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect Mlph-MyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.

The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome-related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow-derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo were found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27-deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live-cell time-lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10-fold increase in granules exhibiting fast movement (>1.5 microm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin-based motility.

The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a-Myrip-MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.

To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation.

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.

A mutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype "chocolate" (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38(G19V) is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.

Two of the major proteins secreted by the prostate epithelium secretory cells are PSA (prostate-specific antigen) and PSAP (prostatic-specific acid phosphatase). The molecules involved in the secretory machinery of PSA and PSAP, and the regulation of this machinery, remain unknown. In the present paper, we provide evidence that JFC1 [synaptotagmin-like protein (slp1)], a Rab27a- and PtdIns(3,4,5)P3-binding protein, regulates the androgen-dependent secretion of PSAP and PSA in human LNCaP prostate carcinoma cells. Androgen-dependent PSAP secretion was significantly inhibited in cells that expressed the C2A domain of JFC1 [PtdIns(3,4,5)P3-binding-domain], but was unaffected by JFC1 overexpression. Conversely, PSA secretion was not inhibited by the C2A domain of JFC1. We show, using immunofluorescence analysis, that JFC1 co-localizes with PSAP, but rarely with PSA, in prostate granules, suggesting that JFC1 is part of the PSAP secretory machinery. However, PSA secretion was significantly increased in LNCaP cells that overexpressed JFC1, indicating that the secretion of PSA is susceptible to variations in the intracellular concentration of JFC1. Both PSAP and PSA secretion was increased by overexpression of wild-type Rab27a or the constitutively active Rab27aQ78L. The secretion of PSA was partially inhibited in the presence of LY294002, while the secretion of PSAP was completely abolished by the PI3K (phosphoinositide 3-kinase) inhibitor. This supports the view that PI3K plays a differential role in the secretion of prostate secretory markers. In conclusion, we present evidence that JFC1 differentially regulates the secretion of PSAP and PSA, and that Rab27a and PI3K play a central role in the exocytosis of prostate-specific markers.

Rab proteins are members of the superfamily of Ras-like small GTPases and are involved in several cellular processes relating to membrane trafficking and organelle mobility throughout the cell. Like other small GTPases, Rab proteins are initially synthesized as soluble proteins and for membrane attachment they require the addition of lipid moiety(ies) to specific residues of their polypeptide chain. Despite their well-documented roles in regulating cellular trafficking, Rab proteins own trafficking is still poorly understood. We still need to elucidate the molecular mechanisms of their recruitment to cellular membranes and the structural determinants for their specific cellular localization. Recent results indicate that Rab cellular targeting might be Rab-dependent, and this paper briefly reviews our current knowledge of this process.

Small GTPases of the Rab family are key regulators of membrane trafficking. Each Rab shows a characteristic subcellular distribution, and may serve as an important determinant of organelle identity. The molecular mechanisms responsible for targeting Rabs to specific intracellular compartments, however, remain poorly understood. The divergent C-terminal hypervariable region was postulated to contain Rab targeting information. We generated a series of hybrid Rab proteins by exchanging the hypervariable domains of Rab1a, Rab2a, Rab5a, Rab7 and Rab27a, and analysed their subcellular localisations. We found that the various hybrid proteins retained their targeting to the parent organelle and were functionally active. We conclude that the hypervariable region does not contain a general Rab targeting signal. Furthermore, we identified other regions within the RabF and RabSF motifs that are required for specific targeting of Rab27a to secretory granules or melanosomes, and Rab5a to endosomes. We observed only partial overlap between targeting-determining regions in the Rab proteins examined, suggesting that Rab recruitment may be complex and at least partially Rab-specific. Mutations in these targeting-determining regions induced localisation to the ER, an observation that further strengthens our previous finding that ER/Golgi membranes serve as the default location for Rabs that have lost targeting information.

Patients with the autosomal recessive Griscelli-Pruniéras syndrome type II are immunologically impaired and have an unusual silvery-grey hypopigmented colour of scalp hair, eyelashes and eyebrows but no noteworthy pigmentary abnormalities of the skin. In most Griscelli patients, the RAB27A gene, which encodes a small GTPase that is associated with the melanosome membrane in melanocytes, is mutated. Here we discuss a genomic RAB27A deletion found in a 21-month-old Moroccan Griscelli patient. Additionally, we provide evidence that the loss of functional Rab27a in melanocytes of this Griscelli patient is partially compensated by the up-regulation of Rab27b, a homologue of Rab27a. By real-time quantitative PCR and western blot analysis, we found that Rab27b mRNA and protein, expressed at low levels in normal human melanocytes, is significantly up-regulated in melanocytes derived from this patient. Our immunofluorescence and yeast two-hybrid screening studies reveal that Rab27b can form a tripartite complex on the melanosome membrane with Melanophilin, a Rab27a effector, and protein products of Myosin Va transcripts that contain exon F. Our data suggest that up-regulated Rab27b in melanocytes of the Griscelli patient can partially take over the function of Rab27a, which could explain the fact that this patient had an evenly pigmented skin and was able to tan.

The remarkable degree of specificity with which Rab GTPases recognise distinct subsets of intracellular membranes forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the right membrane at the right time. The molecular mechanisms controlling Rab localisation, however, have proved tricky issues to address. It is becoming increasingly apparent that multiple factors contribute to the specificity of Rab localisation and the close coordination of membrane targeting with Rab activation. With important new insights into the mode of action of the general Rab regulators REP and RabGDI, as well as the demonstration that novel factors such as Yip3/Pra1 act as GDI displacement factors and that signals within Rab proteins contribute to targeting specificity, a better understanding of the concepts governing Rab recruitment and function is now beginning to emerge. The diversity of cellular processes regulated by Rab family members is made possible, not only by the wide range of effectors they recruit, but also by the different mechanisms regulating their own targeting and activation.

The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles. Some actin-based myosin motors have been implicated in this process, particularly members of the class V myosins. We discuss here the emerging role of the Ras-like GTPases of the Rab family as regulators of myosin function in organelle transport. Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery. Better understood is the case of the melanosome where Rab27a recruits a specific effector called melanophilin, which in turn binds myosin Va. The presence of a linker protein between a Rab and a myosin may represent a general mechanism. We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers. Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

Background/aims: To report the generation of a new mouse model for a genetically determined corneal abnormality that occurred in transgenesis experiments., Methods: Transgenic mice expressing mutant forms of Rab27a, a GTPase that has been implicated in the pathogenesis of choroideremia, were generated., Results: Only one transgenic line (T27aT15) exhibited an unexpected eye phenotype. T27aT15 mice developed corneal opacities, usually unilateral, and cataracts, resulting in some cases in phthisical eyes. Histologically, the corneal stroma was thickened and vacuolated, and both epithelium and endothelium were thinned. The posterior segment of the eye was also affected with abnormal pigmentation, vessel narrowing, and abnormal leakage of dye upon angiography but was histologically normal., Conclusion: Eye abnormality in T27aT15 mice results from random insertional mutagenesis of the transgene as it was only observed in one line. The corneal lesion observed in T27aT15 mice most closely resembles posterior polymorphous corneal dystrophy and might result from the disruption of the equivalent mouse locus.

Vesicular transport is a complex multistep process regulated by distinct Rab GTPases. Here, we show for the first time that an EGFP-Rab fusion protein is fully functional in a mammalian organism. We constructed a PAC-based transgenic mouse, which expresses EGFP-Rab27a under the control of endogenous Rab27a promoter. The EGFP-Rab27a transgene was fully functional and rescued the two major defects of the ashen Rab27a knockout mouse. We achieved cell-specific expression of EGFP-Rab27a, which faithfully followed the pattern of expression of endogenous Rab27a. We found that Rab27a is expressed in an exceptionally broad range of specialized secretory cells, including exocrine (particularly in mucin- and zymogen-secreting cells), endocrine, ovarian, and hematopoietic cells, most of which undergo regulated exocytosis. We suggest that Rab27a acts in concert with Rab3 proteins in most regulated secretory events. The present strategy represents one way in which the complex pattern of expression and function of proteins involved in specialized cell types may be unraveled.

Megakaryocytes release platelets by reorganizing the cytoplasm into proplatelet extensions. Fundamental to this process is the need to coordinate transport of products and organelles in the appropriate abundance to nascent platelets. The importance of the Rab family of small GTPases (guanosine 5'-triphosphatases) in platelet biogenesis is revealed in gunmetal (gm/gm) mice, which show deficient Rab isoprenylation and macrothrombocytopenia with few granules and abnormal megakaryocyte morphology. Although some Rab proteins are implicated in vesicle and organelle transport along microtubules or actin, the role of any Rab protein in platelet biogenesis is unknown. The limited number of Rab proteins with defective membrane association in gm/gm megakaryocytes prominently includes Rab27a and Rab27b. Normal expression of Rab27b is especially increased with terminal megakaryocyte differentiation and dependent on nuclear factor-erythroid 2 (NF-E2), a transcription factor required for thrombopoiesis. Chromatin immunoprecipitation demonstrates recruitment of NF-E2 to the putative Rab27B promoter. Inhibition of endogenous Rab27 function in primary megakaryocytes causes severe quantitative and qualitative defects in proplatelet formation that mimic findings in gm/gm cells. Rab27b localizes to alpha and dense granules in megakaryocytes. These results establish a role for Rab27 in platelet synthesis and suggest that Rab27b in particular may coordinate proplatelet formation with granule transport, possibly by recruiting specific effector pathways.