Phosphatidylinositol 3-kinases and their roles in phagosome maturation

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1 Review Phosphatidylinositol 3-kinases and their roles in phagosome maturation Emily P. Thi* and Neil E. Reiner*,,1 Departments of * Medicine (Division of Infectious Diseases) and Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada RECEIVED FEBRUARY 1, 2012; REVISED APRIL 6, 2012; ACCEPTED APRIL 18, DOI: /jlb Abbreviations: / deficient, Akt protein kinase B, APPL1 adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper/bin-amphiphysin-rvs domain 1, ARF ADP-ribosylation factor, CLR C-type lectin receptor, CORVET class C core vacuoleendosome tethering complex, CR complement receptor, EEA-1 early endosomal antigen 1, GAP GTPase-activating protein, GEF guanine nucleotide exchange factor, GDI guanine nucleotide dissociation inhibitor, HOPS homotypic vacuole fusion and protein-sorting complex, hvps34 human vacuolar protein sorting 34, InsP 3 inositol-1,4,5-trisphosphate, LAMP lysosome-associated membrane protein, LC3-II autophagy protein, ManLAM mannosylated lipoarabinomannan, MBL mannose-binding lectin, Mtb Mycobacterium tuberculosis, Pam 3 Cys tripalmitoyl-s-glyceryl cysteine, PI(3,4)P 2 phosphatidylinositol (3,4)-bisphosphate, PI(3,4,5)P 3 phosphatidylinositol (3,4,5)-trisphosphate, PI(4,5) 2 phosphatidylinositol (4,5)-bisphosphate, PI3/4/5P phosphatidylinositol 3/4/5-phosphate, PIKI- 1 C. elegans Class II PI3K, PTEN phosphatase and tensin homolog, Rab GTPase rat sarcoma analog in brain GTPase, Rac GTPase member of Rho family of rat sarcoma-like GTPases, Ras rat sarcoma, Rho GTPase family of rat sarcoma-like GTPases, RILP Rab-interacting lysosomal protein, SLAMF signaling lymphocyte-activation molecule family, SP-A/ D surfactant, pulmonary-associated protein A/D, Syt synaptotagmin, Trif Toll-IL-1R domain-containing adaptor-inducing IFN-, Vamp7/ Vam7p vesicle-associated membrane protein 7, Vps vacuolar protein sorting ABSTRACT Phagosome maturation is a highly organized and sequential process that results in the formation of a microbicidal phagolysosome. This results in crucial contributions to innate and adaptive immunity through pathogen clearance and antigen presentation. Thus, it is important to understand the regulatory networks that control the extent and nature of phagosome maturation. PI3Ks are lipid kinases that catalyze the phosphorylation of the 3= position of the inositol ring. This enzyme family is divided into three classes based on structure and substrate preferences. Previously, only the class III PI3K, hvps34, was thought to contribute to phagosome maturation. Recent evidence, however, suggests important contributions by class I PI3Ks in bringing about the diverse phagosome maturation phenotypes. Class I PI3Ks have also been implicated in the activation of Rab GTPases that function in maturation, such as Rab14. In addition, recent studies have illuminated the overlap between phagosome maturation and autophagy, which itself is regulated by multiple classes of PI3K. Taken together, a picture of phagosome maturation is emerging in which multiple classes of PI3Ks are involved in modulating maturation phenotypes. This review summarizes the known contributions of PI3Ks to phagosome maturation. Special emphasis is placed on the impact of PI3Ks on different maturation outcomes stemming from the engagement of diverse phagocytic receptors and on Rab and Ca 2 signaling cascades. J. Leukoc. Biol. 92: ; Introduction The ingestion of prey by phagocytic cells leads to the formation of a vacuole referred to as a phagosome. These organelles are by no means static entities. Rather, in the absence of opposing forces, they undergo a dynamic process of phagosome maturation, resulting ultimately in fusion with lysosomes. These mature phagolysosomes are highly destructive antimicrobial environments that play key roles in the innate immune response as well as cellular processes such as autophagy, antigen processing, presentation and others. At its most basic, phagosome maturation is a sequential, fluid process involving interactions of the maturing vacuole with diverse vesicles from the endosomal system. Through these interactions, the phagosomal environment takes on the characteristics of the endosomes with which it has interacted. As such, phagosome maturation stages are defined by the acquisition of protein markers used to characterize specific endosome types [1, 2]. For example, the small GTPase Rab5, the transferrin receptor, and EEA-1 define an early maturation stage, whereas acquisition of Rab7, cathepsin D, and lysosomal membrane proteins such as LAMP-1 and -2, indicates a more advanced, late stage of maturation. Progression through each of these maturation stages is dependent on the interplay between signaling cascades and regulatory proteins involved in the dynamics of vesicular trafficking and membrane fusion. Starting with the receptors used for phagocytosis and up until fusion of phagosomes with lysosomes, PI3Ks and their 1. Correspondence: Division of Infectious Diseases, University of British Columbia, Rm. 452D, 2733 Heather St., Vancouver, BC, Canada, V5Z 3J5. ethan@mail.ubc.ca /12/ Society for Leukocyte Biology Volume 92, September 2012 Journal of Leukocyte Biology 553

2 lipid products play important roles in regulating many aspects of the maturation process. PI3Ks catalyze the phosphorylation of the 3= position on the inositol ring. Three classes of PI3Ks have been identified and grouped according to enzyme structure, substrate specificities, and products formed. The heterodimeric class I PI3Ks are divided into two subgroups; class IA enzymes share one of several common regulatory subunits (p85 and -, p50, and p55 and - ) and different catalytic subunits (p110, -, and - ). The lone class IB enzyme consists of a p101 or p84 regulatory subunit and contains the p110 catalytic subunit [3, 4]. Class I PI3Ks have been shown to catalyze the production of PI(3,4)P 2 and PI(3,4,5)P 3. In contrast, class II PI3Ks lack a regulatory subunit and contain three isoforms (C2, -, and - ). These enzymes catalyze the production of PI3P and PI(3,4)P 2 from PI and PI4P, respectively. The lone class III enzyme is hvps34, which exists as a heterodimer along with its regulatory subunit, p150/vps15. This PI3K produces PI3P from phosphatidylinositol. PI3K activity has been shown to regulate phagosome maturation downstream of diverse phagocytic receptors, and this involves the activation and recruitment of multiple small Rab GTPases that modulate endosomal trafficking. The activities of these small GTPases, part of the Ras GTPase superfamily, play important roles in allowing the maturing phagosome to interact with early and late endosomes and lysosomes. For example, Rab5 is essential for nascent phagosomes to interact with early endosomes [5 7], and the recruitment of Rab7 to phagosomes promotes interactions with late endosome and lysosomes [6, 8, 9]. In addition to Rab activity, Ca 2 -dependent signaling cascades are needed to ensure that maturation proceeds toward the formation of a degradative compartment [10 12]. Like the Rabs, certain Ca 2 -dependent signaling events depend on the function of PI3Ks, which serve to link effector proteins activated by Ca 2 to the phagosome membrane through the formation of lipid effectors. The contributions of the specific PI3K classes to phagosome maturation initiated downstream of distinct phagocytic receptors, and how these lipid kinases regulate the activities of the Rab GTPases and Ca 2 signaling cascades involved in the maturation process, are the focus of this review. PART I. REGULATION OF PHAGOSOME MATURATION VARIES ACCORDING TO THE PHAGOCYTIC RECEPTOR ENGAGED; ROLES OF PI3Ks Uptake of prey by a cell involves the engagement of phagocytic receptors located on the plasma membrane. Two broad groups of phagocytic receptors have been characterized (summarized in Table 1). Opsonic receptors include the antibodybinding Fc R, which serves to mediate the uptake of prey opsonized with Ig, and the CRs CR1 CR4, which initiate the uptake of prey coated with C3b, ic3b, and C4b (for a review, see ref. [21]). Nonopsonic receptors include CLRs, scavenger receptors, and other PRRs such as TLRs and the microbial sensing SLAMF [20, 22, 23]. Once ingested, maturation of the newly formed vacuole is influenced by the particular phagocytic receptors that were engaged. An excellent example of how phagosome maturation can differ according to the receptors used for ingestion was provided by a study that examined maturation of phagosome containing the human pathogen Mtb. Upon uptake by a macrophage in the absence of added opsonins, Mtb blocks maturation at a stage before phagolysosome generation [1]. In striking contrast, however, IgG opsonization of virulent Mtb and uptake via Fc R vitiates the ability of Mtb to bring about phagosome maturation arrest [24]. These findings indicate that: (1) the ability of Mtb to block phagosome maturation requires that its uptake is independent of Fc R, and of more general importance to cell biology, that (2) the dynamics of phagosome maturation are dependent on the phagocytic receptors involved. Subsequent detailed studies examining the effects of specific ligand-receptor interactions on phagosome maturation [25, 26] have further clarified the mechanisms that regulate this dynamic process. Below, we address each type of the phagocytic receptors in turn by examining firstly, the evidence that uptake by each of these receptors results in different phagosome maturation out- Receptors Opsonic receptors Fc R CRs Collectins TABLE 1. Roles of PI3Ks in Phagosome Maturation, According to Specific Receptors PI3K involvement PI(3,4,5)P 3 needed for phagocytic cup closure [13]; PI3P needed for phagosome maturation PI(3,4,5)P 3 needed for phagocytic cup closure [13]; PI(3,4,5)P 3 needed for actin tail formation [14] PI(3,4,5)P 3 activation of PKC, leading to up-regulation of Rab7 expression [15]; activation of PKC effects on actin cytoskeleton through Rac GTPases Nonopsonic receptors CLRs Dectin 1 uptake of Candida albicans results in phagosomes with PI3P, PI(3,4)P 2, PI(3,4,5)P 3, PI(4,5)P 2, and actin polymerization [16] Nonphagocytic PRRs TLRs SLAMF TLR-2 and -4 signaling leading to autophagy induction via Beclin1/hVps34 [17]; enhanced PI3P levels on phagosomes containing zymosan [17]; MyD88 and Trif interact with Beclin1 [18]; TLR-4 signaling results in increased hvps34 membrane association [19] Recruits Beclin/hVps34 complex to phagosome, leading to PI3P production and enhanced maturation [20] 554 Journal of Leukocyte Biology Volume 92, September

3 Thi and Reiner PI3Ks and phagosome maturation comes, and secondly, whether PI3K activity is implicated in bringing about these outcomes. Opsonic receptors Fc R-mediated phagocytosis. There are three classes of human Fc Rs [Fc RI or CD64, Fc RIIA and -B (CD32), and Fc RIIIA (CD16a) and -B (CD16b)]. Of the three classes of Fc Rs, Fc RI, Fc RIIA, and Fc RIII act as activation receptors, which require prey coated with multiple IgG molecules in an immune complex to bind and initiate phagocytosis [27 30]. In monocytes and macrophages, Fc RI and -III but not Fc RIIA must also associate with a subunit to be expressed at the cell surface [31] (for a review of Fc R structure and function, see ref. [32]). The ITAM present in the subunit allows the initiation of signaling cascades upon receptor clustering on the cell surface [33]. However, association of ITAM-containing receptors with Fc RIIB, a member of the immune inhibitory receptor family, results in tyrosine phosphorylation within the inhibitory ITIM motif present in Fc RIIB. This in turn results in the recruitment of the inositol phosphatase SHIP and inhibition of activation (for a review, see ref. [34]). Within the ITAMs, initial phosphorylation of the tyrosine residues by the Src family of tyrosine kinases allows Src homology 2 domain-mediated recruitment of the tyrosine kinase Syk, which then phosphorylates tyrosine residues within adjacent ITAMs present in the Fc R cluster [35 37]. Class IA PI3Ks are activated following Fc R clustering [38, 39], and the activity of these PI3Ks is needed for closure of phagosomes containing particles 3 m in diameter [40, 41]. PI(3,4,5)P 3 has been shown to accumulate on the phagocytic cup and to disappear quickly upon closure [13]. The presence of PI(3,4,5)P 3 promotes pseudopod extension and the contraction event required for phagosome closure through its recruitment of the unconventional pleckstrin homology domain-containing protein myosin X [41 43]. In addition, PI(3,4,5)P 3 mediates the signal transmission events which occur between Rho GTPases located at the phagocytic cup. It does this by promoting deactivation of the Rho GTPases Cdc42 and ARF6, which are involved in actin polymerization during early stages of phagocytosis. In parallel to this, PI(3,4,5)P 3 also brings about the activation of late-stage Rac2 and ARF1, which function in phagosome closure [44 46]. PI(3,4,5)P 3 -mediated deactivation of Cdc42 is thought to be necessary to allow polymerized actin around the nascent phagosome to dissipate, thus allowing phagosome interactions with the endocytic system [40, 47]. At this stage of maturation, PI3P first appears on the vacuole due to the activity of the class III enzyme hvps34 [48], although conceivably, PI3P can also be formed via the action of lipid phosphatases on PI(3,4,5)P 3. The 3=-inositol phosphatase PTEN has been implicated in the breakdown of phagosomal PI(3,4,5)P 3, as its overexpression abrogates uptake of IgG-opsonized sheep red blood cells [49]. However, PTEN does not appear to be recruited to the nascent phagosome and is thus thought to exert its effects in a global manner [50]. It is known that the 5= phosphoinositide phosphatase SHIP1 is recruited to the nascent phagosome at a time when PI(3,4,5)P 3 levels drop dramatically, and its presence is thought to contribute to the disappearance of PI(3,4,5)P 3 upon phagocytic cup closure [13]. The PI(3,4)P 2 formed as the result of SHIP1 activity could then be degraded further by the activity of PI4Ps, resulting in PI3P formation on the phagosome membrane independent of hvps34. Rab5 has been demonstrated to interact with the class IA PI3K p110, as well as with hvps34 in vitro [51]. Active Rab5 has also been shown to interact with PI-4- and -5-phosphatases in vitro, and this may lead to PI3P production from PI(3,4,5)P 3 on early endosomes [52]. Whether the presence of Rab5 on phagosomes also recruits PI-4- and -5-phosphatases to form a pool of PI3P derived from PI(3,4,5)P 3 has not yet been demonstrated. However, distinct from the phagocytic cup, no evidence has been found for PI(3,4,5)P 3 on completed phagosomes formed around IgG-coated beads, although class I PI3Ks are recruited to these phagosomes [14]. The failure to detect PI(3,4,5)P 3 on these completed phagosomes may reflect the lack of PI(4,5)P 2 substrate resulting from PLC-mediated degradation [53 55]. In addition, recent work from the laboratory of Sergio Grinstein [56] has identified the Rab5 effectors and inositol 5-phosphatases oculocerebrorenal syndrome of Lowe protein (OCRL) and 75kDa inositol polyphosphate 5-phosphatase B (Inpp5B), as being involved in PI(4,5)P 2 removal from the sealing phagosome. In addition to any potential role for class I PI3Ks in contributing to phagosomal PI3P levels, a recent report studying apoptotic corpse removal in the nematode Caenorhabditis elegans implicated the class II PI3K, PIKI-1, in forming PI3P seen on nascent phagosomes, with Vps34 responsible for PI3P production at later stages of maturation [57]. Whether class II PI3Ks also contribute to phagosomal PI3P production in mammalian cells remains to be determined. Studies done with Fc RIIA-transfected p85 and - doubleknockout fibroblasts indicate that hvps34 plays a major role in phagosome maturation downstream of Fc R phagocytosis by allowing the recruitment of the Rab5 effector, EEA-1 [58 60]. hvps34 and its adaptor protein Vps15/p150 have also been demonstrated to interact with Rab7, thus promoting subsequent phagolysosome formation [48, 61]. In this model system, the class I PI3Ks were found to be required for mediating formation and closure of the phagocytic cup and not maturation [48]. However, the contributions of PI3K to phagosome maturation, particularly with regards to phagolysosome fusion, likely involve more than just simply mediating the recruitment of Rab5 effectors and Rab7. This inference is based on several related findings. For instance, wortmannin-treated murine macrophages (RAW264.7)fed IgG-coated latex beads showed an 50% reduction in Rab7 recruitment to phagosomes, but this Rab7 was still in an active state and was able to recruit the downstream effector RILP [62]. Furthermore, wortmannin treatment of phagocytic cells routinely disrupts phagolysosome fusion by upwards of 90%. It seems unlikely then that a 50% decrease in Rab7 phagosomal recruitment alone could account for this dramatic phenotype of maturation arrest [62]. CR-mediated phagocytosis. In contrast to Fc R signaling, tyrosine kinase activity seems not to be involved downstream of CR engagement, as treatment of cells with the broad-range tyrosine kinase inhibitor herbimycin A had no effect on CR phagocytosis [25, 63]. However, this issue is unresolved, as there has been a recent report of tyrosine kinase activity downstream of 2 integrin clustering leading to PI3K activation Volume 92, September 2012 Journal of Leukocyte Biology 555

4 [64]. Regardless, as is the case for Fc R phagocytosis, both PI(3,4)P 2 and PI(3,4,5)P 3 appear on the phagocytic cup during integrin (CR)-mediated phagocytosis, where they function to trigger Ca 2 signaling to promote cup closure [65]. Ca 2 release may result from activation of phosphoinositide-specific phospholipase C (PI-PLC), which hydrolyzes PI(4,5)P 2 to initiate an InsP 3 -mediated release of Ca 2 from ER stores (for an in-depth review of Ca 2 release and effects on phagosomes, see ref. [66]). In addition to phagocytic cup closure, Ca 2 triggers downstream events that are needed for phagosome maturation. The role that PI3Ks play in aiding Ca 2 effects on maturation is discussed further below and is summarized in Fig. 1. Unlike phagosomes formed after Fc R engagement, CR3 phagosomes display a second wave of PI(3,4,5)P 3 formation after phagosome cup closure. This, along with the presence of PI3P, leads to the formation of actin tails that propel phagosomes through the cell [14]. The consequences of this propulsion of CR3 phagosomes are unknown, although the increased motility of these vesicles may hinder their interactions with endosomes, thereby causing a delay in phagosome maturation [14]. Such a delay may promote the survival of intracellular pathogens such as Mtb, which is taken up primarily through CR3 [67, 68], and Mycobacterium leprae, which enters macrophages via CR3 and CR1 [69]. Figure 1. Proposed involvement of PI3Ks in Ca 2 signaling cascades required for phagosome maturation. Phagocytosis of prey triggers sphingosine kinase activation by an unknown mechanism. Sphingosine kinase can be activated at the plasma membrane or on the phagosome. This leads to the production of sphingosine 1-phosphate (S1P), which can then activate the PLC pathway, resulting in the generation of InsP 3. InsP 3 then mediates the release of Ca 2 stores through its receptors in the ER membrane. Alternatively, S1P may directly activate the release of Ca 2 through binding to an unidentified ER receptor. The release of Ca 2 results in a localized, transient increase in the concentration of this cation, which triggers the activation of calmodulin. Calmodulin recruits hvps34 to the phagosome membrane, leading to the production of PI3P that is necessary for recruitment of the SNAREs syntaxin 6 and Vamp7. In addition, the increase in cytosolic Ca 2 can trigger the activation of Syts, which may be able to bind to the vacuole through PI(3,4,5)P 3 generated by class I PI3Ks on the phagosome membrane. Syts may aid in the priming of SNAREs for membrane fusion in a similar manner to that seen in SNARE- mediated exocytosis, although this has yet to be experimentally determined. 556 Journal of Leukocyte Biology Volume 92, September

5 Thi and Reiner PI3Ks and phagosome maturation Influence of collectins on phagosome maturation. In addition to IgG and complement, collectins have been shown to contribute to both phagocytosis and phagosome maturation. Collectins are soluble PRRs belonging to the C-type lectin superfamily [70 72] (see Table 2 for a summary of collectins). One key example is MBL, a soluble opsonin which recognizes mannose and N-acetylglucosamine sugars on microbial surfaces [82 84]. Although well-known for its role in activating the complement cascade (for a review, see refs. [85, 86]), MBL has also been shown to enhance phagocytosis of Salmonella enterica [87] and M. avium [88], likely through uptake via CR1 and calreticulin/cd91 receptors [89, 90]. In addition to promoting uptake of prey, there is evidence that MBL can enhance the maturation of the resulting phagosomes. For example, MBL-enhanced uptake of N. meningitidis augments the acquisition of LAMP-1 on phagosomes [74]. The pulmonary surfactant proteins SP-A and SP-D are collectins that play key roles in innate immunity within the lung by opsonizing and mediating the agglutination of bacteria [91]. SP-D appears to enhance the fusion of phagosomes containing Mtb with lysosomes, as assessed by increased colocalization of Mtb phagosomes with the lysosomal membrane proteins CD63 and LAMP-1 [79]. As SP-D can bind to the mannose caps of Mtb ManLAM, it is thought that the enhanced vacuole maturation seen with SP-D may result from it masking the mannose caps of ManLAM. This would prevent Mtb uptake through mannose receptors, which is unfavorable for phagosome maturation [92], and potentially redirect uptake to other more favorable phagocytic receptors. SP-A and SP-D have also been shown to promote maturation of L. pneumophilia-containing phagosomes to acidic, LAMP-1-positive compartments [78]. In addition, SP-A has been found to enhance phagolysosome fusion in alveolar macrophages through up-regulation of cellular expression of Rab7, and this was dependent on PI3K/Akt activation [15]. Under these conditions, coimmunoprecipitation experiments indicated that Rab7 interacted with atypical PKC [15]. This is of particular interest, as previous studies had implicated PI(3,4,5)P 3 in activating PKC [93, 94], and this kinase has been shown to regulate actin cytoskeleton rearrangements by Rac GTPases [95, 96]. Taken together, this evidence suggests that pulmonary surfactant proteins are involved in modulating phagosome maturation, and this is dynamically regulated by class I PI3K activity. NONOPSONIC RECEPTORS PRRs CLRs. The opportunistic fungal pathogen, Candida albicans, enters macrophages using the CLR Dectin-1 [97 99], after which it resides in a phagolysosome and resists killing [100]. Early phagocytic signaling involving Src and Syk tyrosine kinase family activation during Dectin-1-mediated uptake of C. albicans mimics that which occurs during Fc R phagocytosis, and this is largely a result of the presence of an ITAM-like motif in the cytoplasmic tail of Dectin-1 [101, 102]. A study examining late-stage phagosome maturation of C. albicans-containing phagosomes noted extensive and localized actin polymerization on the vacuole, which was accompanied by the presence of PI3P, PI(3,4)P 2, PI(3,4,5)P 3, and PI(4,5)P 2 [16]. The late stage appearance of these phosphatidylinositols on the phagosome membrane, along with polymerized actin, bears a striking similarity to that seen on phagosomes formed from CR3-mediated uptake [14]. However, treatment of cells infected with C. albicans with the pan-pi3k inhibitor LY did not show any effects on phagosome actin polymerization, leading the authors to conclude that this was PI3K-independent [16]. The consequences of the appearance of these phosphatidylinositol species in terms of promoting phagosome-lysosome interactions were not assessed in the C. albicans or CR phagosome actin studies, although the formation of actin tails in CR3 phagosomes is thought to delay maturation by promoting phagosome movement away from the proximity of lysosomes [14]. The comparatively less mobile C. albicans phagosomes, on the other hand, displayed rapid phagolysosomal fusion [103], even above that seen with Fc R phagosomes [104]. Further work examining the biological significance of phosphatidylinositol formation on late-stage phagosomes formed from CR- and Dectin-1-mediated uptake will provide interesting clues as to the roles PI3Ks play in regulating maturation at the junction of phagosomes and lysosomes. TABLE 2. Collectins and Their Roles in Phagocytosis and Phagosome Maturation Collectin MBL Pulmonary surfactant proteins SP-A SP-D Function Increases uptake of Staphylococcus aureus and apoptotic cells and is trafficked to phagosome with prey [73]; increases uptake of Neisseria meningitidis and enhances EEA-1 and LAMP- 1 acquisition [74]; binding and phagocytosis of nucleic acid ligands [75] Stimulates phagocytosis of Klebsiella pneumoniae [76]; opsonization of Francisella novicida increases bacteria intracellular survival [77]; increases colocalization of Legionella pneumophila phagosomes with LAMP-1 [78]; enhances Escherichia coli lysosomal delivery by up-regulating Rab7 expression and activation via PKC /PI3K/Akt signaling [15] Reduces uptake of Mtb, decreases bacterial intracellular survival, promotes phagolysosome fusion [79]; increases colocalization of L. pneumophila phagosomes with LAMP-1 [78]; up-regulates phagocytosis of Mycobacterium avium [80]; enhances phagocytosis of Cryptococcus neoformans and increased intracellular survival [81] Volume 92, September 2012 Journal of Leukocyte Biology 557

6 NONPHAGOCYTIC PRRS THAT INFLUENCE PHAGOSOME MATURATION TLRs and phagosome maturation The role of TLRs in influencing phagosome maturation has been a subject of much study and debate. MyD88 / mice have been shown to have defects in phagocytosis and phagosome maturation [22, 23, ], and stimulation of TLR-4 signaling during apoptotic cell phagocytosis appears to delay lysosomal marker acquisition [108]. Blander and Medzhitov [22] posited that TLRs are involved in stimulating phagocytosis and promoting phagosome maturation, as bone marrow-derived macrophages from MyD88 / and TLR-2 and -4 double-knockout mice fed bacterial prey displayed reduced phagocytic uptake and vacuole maturation when compared with WT cells. However, this work attributed the phenotype to TLR stimulation solely on the basis of the genotypes of the macrophages studied. In contrast, work done by Yates and Russell [23] suggested that TLR stimulation does not influence phagosome acidification or the extent of phagolysosomal fusion. This study used silica beads opsonized with mannose or IgG which were then coated with the TLR-2 and -4 ligands Pam 3 Cys and LPS, respectively. Using carboxyfluorescein or Oregon Green to measure phagosomal ph and a fluorescence resonance energy transfer-based method to determine the extent of phagolysosome interactions, no differences were observed using macrophages isolated from WT or TLR-2 / mice that were fed either control or ligand-coated beads [23]. While this suggested the conclusion that neither TLR-2 nor TLR-4 engagement influences maturation, mannose or IgG opsonization as done would have introduced a confounding variable. It is possible that uptake via these specific phagocytic receptors may have influenced phagosome maturation to such an extent that additional TLR stimulation would have had little impact. Thus, it may be premature to conclude that TLRs do not influence phagosome maturation. Indeed, subsequent, more detailed characterization of phagosome functionality in terms of assessing hydrolytic activity of proteases, lipases, and glycosidases suggest that TLRs may well influence different aspects of phagosome maturation in subtle ways [109]. Supporting this, TLR-2 was recently shown to enhance the maturation of M. avium phagosomes in mouse macrophages [110]. In contrast, blocking TLR-2 in M. avium paratuberculosis infected bovine monocytes resulted in increased phagosome acidification and phagolysosome fusion, indicating that TLR-2 in these cells contributed to phagosome maturation inhibition [111]. These apparently conflicting findings about the effect of TLR-2 on phagosome maturation may result from the different mycobacterial and host animal species used. In addition to M. avium paratuberculosis, the latter study had also examined TLR-2 effects on M. avium avium-infected cells and saw no effects on phagosome maturation in this case [111]. It was suggested that this null phenotype might be attributed to the inability of this bacterial strain to cause disease in bovines [111]. In addition, S. enterica serovar typhimurium containing phagosomes were recently shown to be dependent on TLR signaling to enhance acidification of the salmonella-containing vacuole, which triggers the induction of the salmonella pathogenicity island 2 locus and the expression of virulence effectors necessary for bacterial intraphagosomal replication [112]. Thus, it is becoming clear that to some extent and in various ways, TLRs are involved in modulating phagosome maturation. Relevant to this discussion is the question of whether PI3Ks play any roles in mediating any TLR effects on maturation. It has been shown that the intracellular pathogen Trypanosoma cruzi activates Rab5 through a TLR-2-dependent mechanism, and this requires PI3K activity [113]. Moreover, accumulating evidence points to a role of TLRs in promoting autophagy/autophagosome maturation dependent on PI3K activity. Phagosomes containing latex beads coated with Pam 3 Cys rapidly recruited the autophagy marker LC3- II, a phenomenon not observed in the absence of the TLR-2 ligand [17]. LC3-II recruitment to these phagosomes was dependent on PI3K activity, and higher levels of PI3P were observed around phagosomes with TLR stimulation [17]. This PI3P increase also coincided with enhanced acidification of phagosomes containing Pam 3 Cys beads when compared to beads with no ligand, indicating that TLR signaling enhanced phagosome maturation through PI3K activity [17]. Later investigations demonstrated that the TLR adaptor proteins MyD88 and Trif interacted with Beclin1 to induce autophagy [18]. As Beclin1 is a key component of the hvps34 complex that initiates autophagosome formation, one would expect that its association with MyD88 and Trif would also indicate TLR effects on hvps34 activity, subcellular localization, or both. This is supported by a study that showed that LPS treatment increased the presence of hvps34 in cellular membrane fractions [19], which correlates with the enhanced PI3P levels seen on phagosomes containing TLR ligands [17]. All of these studies provide insight into how TLR signaling leading to PI3K activity could enhance phagosome/autophagosome maturation, and doubtless further discoveries will soon come in this active area of investigation. SLAMFamily The receptors for the SLAMfamily serve as costimulatory molecules for adaptive immunity [114]. One member of this family, SLAMF1 (CD150), also appears to play a role in promoting the maturation of phagosomes containing gramnegative bacteria [20]. SLAMF1 interacts with E. coli at the cell surface and is taken up with bacteria into phagosomes, where it up-regulates maturation and NADPH oxidase complex 2 activity. This has been shown to involve recruitment of the hvps34/vps15/beclin-1 complex, leading to increased local accumulation of PI3P [20]. This finding was significant, first, as it indicated the contribution of nonphagocytic receptors to phagosome maturation, and second, it showed that additional, novel pathways exist by which PI3K activity could be modulated to promote maturation from receptor signaling within the phagosome. These findings raise additional questions, such as do other mem- 558 Journal of Leukocyte Biology Volume 92, September

7 Thi and Reiner PI3Ks and phagosome maturation bers of the SLAMfamily regulate phagosome maturation, and if so, how does this compare with SLAMF1? Do different SLAMs synergize with each other in a manner similiar to TLRs? Given that the hvps34/vps15/beclin-1 complex has also been shown to be involved in TLR induction of autophagy [17 19], do SLAMs also synergize with TLR signaling to bring about autophagosome formation and enhanced maturation? Future work with this family of microbial sensors will surely lead to answers to these questions and shed additional light on the roles they play in innate immunity. PART II. REGULATION OF Rab GTPases AND Ca 2 SIGNALING BY PI3K INFLUENCES PHAGOSOME MATURATION Rab GTPase activation and recruitment of effectors Rabs are the largest subfamily of the Ras superfamily of GTPases, and include over 60 proteins that are involved in the regulation of endosomal and exocytic traffic (for detailed reviews of Rab structure and function, please see refs. [115, 116]). Rabs function as molecular switches, being in an active state when bound to GTP and inactive when GTP is hydrolyzed to GDP upon activation of their intrinsic GTPase activity by a GAP [117]. This cycling between active and inactive states is essential to Rab function and enables the attachment of active Rab proteins to the phagosomal membrane via C-terminal lipid geranylgeranyl tails [118]. Inactive, GDP-bound Rabs are cytosolic and complexed with GDIs. Although many models have been proposed to explain the mechanism of Rab activation and membrane attachment, a recent study that examined Rab membrane targeting using semisynthetic fluorescent Rab probes, supports a model in which spontaneous Rab dissociation from GDIs and subsequent replacement of GDP with GTP by GEFs allows membrane attachment. GTP-bound Rab is unable to be bound by the Rab GDI, thereby favoring membrane attachment and allowing active Rabs to migrate to the phagosome membrane [119]. In addition to the importance of Rab GEFs in activating Rab activity, the phosphoinositides PI(4,5)P 2 and PI(3,4,5)P 3 may also contribute to Rab membrane targeting. These negatively charged phosphoinositides are able to bind to the C-terminal polybasic clusters that are present in some Rabs. [120]. Following membrane recruitment, subsequent GAP activation of Rab GTPase activity brings about the conversion of GTP to GDP. This results in binding of the GDI to Rab-GDP, dissociation from the phagosomal membrane, and the completion of a cycle. Figure 2 summarizes the contributions of PI3K to Rab-mediated phagosome maturation, described below. Early phagosomal Rab5 recruitment and activation. Rab5 mediates the fusion of nascent phagosomes with early endosomes. Although Rab5 has been shown to interact with hvps34 [51], phagosomal acquisition of Rab5 can occur independently of PI3K, as wortmannin treatment during the course of Fc R phagosome maturation did not inhibit Rab5 (or late phagosomal Rab7) recruitment [62]. This study did note, however, that inhibition of PI3K activity resulted in prolonged retention of Rab5 on phagosomes [62]. This is particularly interesting, given that several studies have found that the p85 regulatory subunit of class IA PI3Ks displays GAP activity toward Rab5 (in addition to Rab4, Cdc42, and Rac1) [ ], and p85 has been shown to be present on early-to-late phagosomes during Fc R and CR3-mediated phagosome maturation [14]. It may be that the presence of the class IA PI3K p85 regulatory subunit on the phagosome is necessary for Rab5 dissociation from the membrane. However, as wortmannin acts by irreversibly binding to the ATP-binding pocket of the p110 catalytic subunit [124, 125], the inhibitor should not, in principle, affect the GAP activity of p85, which would be free to exert its effects on Rab5. Thus, if p85 were indeed a GAP for Rab5, wortmannin-treated cells should presumably show normal Rab5 displacement from the phagosome. One explanation for this paradox would be an allosteric effect by wortmannin on p85 GAP activity, resulting from p110 catalytic inhibition. Alternatively, it may be that wortmannin, in addition to inhibiting p110 catalytic activity, also affects phagosomal recruitment of the p85 subunit. This would explain the retention of phagosomal Rab5 seen in wortmannin-treated cells [62]. Further experiments to directly test p85 recruitment in wortmannin-treated cells should help to clarify the biological relevance of p85 GAP activity in the context of phagosome maturation. Notwithstanding any contribution from class IA PI3Ks, the class III enzyme hvps34 is a well-established Rab5 effector [48, 51]. This is a critical function, as PI3P is needed for phagosomal recruitment of EEA-1, which serves as a tethering factor to promote fusion of early endosomes with the phagosome [58, 59, ]. Late phagosomal Rab7 and its effectors. During the course of maturation, Rab5 is subsequently replaced on the phagosome membrane by Rab7, in a process termed Rab conversion [130]. The mechanism for this conversion event has been elucidated recently in yeast and in the nematode worm C. elegans. It is initiated by CORVET binding to active Rab5 [131], followed by the recruitment of Mon1/Ccz1 (or SAND-1 in C. elegans) [132]. SAND-1/Mon1 then acts as a GEF for Rab7, mediating its membrane binding [ ], and this is followed by a conversion from CORVET to the HOPS complex [131]. As the multisubunit CORVET and HOPS complexes share four of the same class C Vps proteins (Vps11, Vps16, Vps18, and Vps33), conversion of CORVET to HOPS only requires exchange of the CORVET Vps3 and Vps8 with the HOPS components Vps39 and Vps41 [131]. Both Vps39 and Vps41 have been shown to bind Rab7, with Vps41 binding specifically to Rab7-GTP [135]. By binding to active Rab7 and SNAREs, HOPS serves to bridge Rab7 activation with the SNARE complex primed in its proper orientation (see below) [135]. In addition to Rab7 activity, HOPS membrane recruitment may be dependent on phosphatidylinositides, as purified, active HOPS can bind to PI(4,5)P 2, PI(3,5)P 2, and PI4P on liposomes [136]. The SNAREs that are recruited via HOPS are membranebound proteins that drive fusion events by interacting in trans on opposite membranes. Late endosome/lysosome membrane fusion requires the assembly of a SNARE complex consisting of three Q-SNAREs on one membrane (containing glutaminyl residues essential for fusion) and one R-SNARE (containing an Volume 92, September 2012 Journal of Leukocyte Biology 559

8 Figure 2. Overview of the roles of PI3Ks in the regulation of phagosome maturation by Rabs. (A) Production of PI(3,4,5)P 3 by class IA PI3K enables the recruitment of Akt to the phagosome. The latter inactivates its substrate, AS160, which reduces its efficacy as a Rab14 GAP. This allows Rab14 to be recruited to the phagosome, where it mediates the interactions of the phagosome with early endosomes (EE). Subsequent dephosphorylation of PI(3,4,5)P 3 by the inositol 5-phosphatase APPL1 results in inactivation of Akt. This releases the inhibition of AS160, thereby allowing it to exert its GAP function on Rab14, resulting in Rab14 dissociation from the phagosome membrane. This is necessary for phagosome interactions with late endosomes (LE) or lysosomes (LYS). (B) hvps34 is recruited to the early phagosome membrane, where it produces PI3P. Rab5 recruitment, together with PI3P, then promotes acquisition of EEA1, which serves as a tethering factor for phagosome interactions with early endosomes. A rab conversion event then takes place, which results in the replacement of Rab5 with Rab7 on the phagosome membrane. Rab7 then recruits its effector proteins, RILP and the HOPS complex, both of which are necessary for interactions of late endosomes and lysosomes with the maturing vacuole. arginyl residue) on the opposing membrane. HOPS has been shown to proofread the assembled SNAREs to ensure their correct formation into a 3Q 1R complex [137]. In addition to HOPS tethering, PI(4,5)P 2 and PI3P are essential for enhancing SNARE-mediated membrane fusion [ ]. PI3P is specifically required for membrane targeting of the soluble SNARE Vam7p, which contains a phox homology domain that binds to PI3P [141, 142]. These studies and others suggest that in addition to active Rab7, PI3K activity is needed for proper targeting of HOPS and SNAREs required for phagolysosome fusion. Other Rab contributions to phagosome maturation. In addition to Rab5 and Rab7, other Rab GTPases have been implicated in regulating phagosome maturation. Rab14 and Rab2, for example, have been shown to act redundantly in phagosome maturation by recruiting lysosomes to phagosomes containing apoptotic cell corpses in C. elegans [143]. Recruitment of Rab14 and Rab2 themselves is dependent on Rab5 and PI3P, as loss of Rab5 or Vps34 function abrogated Rab2 and Rab14 recruitment to phagosomes [143]. More evidence for a role for Rab14 in regulating phagosome maturation was shown in a study that found that its retention on phagosomes contributed to maturation arrest of the Mtb vacuole. This study attributed Rab14 regulation of phagosome maturation to the persistent presence of Rab14 promoting prolonged early endosome-phagosome interactions, thus retarding maturation [144]. This view of Rab14 function that of promoting early endosome-phagosome interactions, as opposed to having a 560 Journal of Leukocyte Biology Volume 92, September

9 Thi and Reiner PI3Ks and phagosome maturation role in mediating phagolysosome fusion is more likely to be correct, as numerous studies have implicated Rab14 as having a role in promoting biosynthetic/recycling sorting between the Golgi and early endosomes in mammalian cells [ ]. However, RabD, the Rab14 homolog in Dictyostelium discoideum, appears to regulate homotypic phagosome and lysosome interactions in this single-cell eukaryote, suggesting that this Rab may participate in different areas of the endosomal network, depending on the model system being investigated [148]. As is the case for Rab5 and Rab7, Rab14 function is also regulated by PI3K. PI3K-induced activation of Akt has been shown to bring about phosphorylation of the Akt substrate and Rab GAP AS160 [149]. This results in the inactivation of AS160 and prevents it from binding to membranes [150]. As AS160 is the GAP for Rab14 [150], inactivation of the former prolongs the activation state of Rab14 and its retention on phagosomes, thereby contributing to phagosome maturation arrest. In support of this role for Akt and AS160 in preventing effective phagosome maturation and the development of an optimal microbicidal vacuole, it was found that Akt inhibitors reduced the intracellular survival of Salmonella typhimurium. It was inferred from this result that by preventing Akt-mediated inactivation of AS160, dissociation of phagosomal Rab14 was no longer impaired, and phagosome maturation leading to fusion with lysosomes was able to proceed [151]. Although this study did not observe Akt on S. typhimurium phagosomes [151], Akt has been found on EEA-1-positive endosomes [152] and Fc R phagosomes [56]. Akt on Fc R phagosomes is inhibited by recruitment of the Rab5 effector and Akt interactor, APPL1, which inhibits Akt activity by promoting the recruitment of inositol 5- phosphatases [56]. Although the latter study did not examine the consequences of Akt phagosomal recruitment and deactivation, it seems plausible that active phagosomal Akt may promote and prolong Rab14-mediated early endosomephagosome interactions by inactivating Rab14 GAP AS160. Conversely, the coordinate or sequential inactivation of Akt by APPL1 via inositol 5-phosphatase removal of the class I PI3K substrate PI(4,5)P 2 might be expected to release AS160 from Akt-mediated inhibition, thereby promoting Rab14 dissociation from the phagosome membrane and progression to phagolysosome fusion (illustrated in Fig. 2A). Future study of the roles that Rab14 and PI3Ks play in phagosome maturation may clarify this picture and suggest contributions by other Rabs to this process. Ca 2 and PI3K signaling combine to influence phagosome maturation A rise in cytosolic Ca 2 levels and subsequent initiation of calmodulin signaling cascades has been shown to be important in allowing phagolysosome formation to occur [10, ]. Although a rise in cytosolic Ca 2 has been characterized extensively upon phagocytosis, there has been a dearth of evidence for Ca 2 oscillations during the late phagosome maturation stage, which would be expected if Ca 2 does indeed influence maturation. However, recent work has suggested the importance of voltage-gated sodium channels such as the macrophage NaV1.5 channel, found on late endosomes [156], in triggering local periphagosomal calcium oscillations which contribute to phagosome maturation. NaV1.5 has been shown to cooperate with the mitochondrial Na /Ca 2 exchanger to bring about periphagosomal calcium oscillations during maturation of bacillus Calmette-Guerin-containing phagosomes [157]. It is expected that future studies, using detailed and precise measurements for intracellular Ca 2 levels during the course of phagosome maturation, will allow further clarification as to whether Ca 2 is involved in regulating maturation. To date, much of what we know about the potential role of PI3K in Ca 2 signaling during phagosome maturation comes from studies examining how the Mtb lipid ManLAM imparts a maturation block in Mtb-infected cells. ManLAM inhibits the delivery of immature cathepsin D from the TGN to phagosomes containing ManLAM-coated beads [158]. It does this, in part, by preventing syntaxin 6 recruitment to phagosomes [158]. Syntaxin 6 is part of the SNARE machinery involved in vesicular trafficking from the TGN to endosomes [159, 160], and its recruitment to phagosomes is PI3K-dependent [158]. Further investigation revealed that the mechanism behind reduced syntaxin 6 recruitment involved the ability of ManLAM to inhibit Ca 2 release and secondarily prevent phagosomal recruitment of PI3K [161, 162]. This conclusion is consistent with a model in which Ca 2 release is required to trigger calmodulin signaling and the recruitment of a hvps34 complex to the phagosome [163]. The resulting PI3P formed may then mediate the phagosomal recruitment of syntaxin 6, thus allowing delivery of lysosomal components from the TGN to the phagosome [158]. These proposed interactions of Ca 2 and PI3K signaling events needed for phagosome maturation are illustrated in Fig. 1. In addition to calmodulin effects, Ca 2 is also detected by the calcium sensors Syts. Although most extensively characterized in the field of neurotransmitter release and exocytosis, these proteins have also recently been shown to contribute to phagosome formation by way of focal exocytosis of endosomal membrane, as well as to maturation of the vacuole itself. Syt V contributes to phagocytosis and delivery of the vacuolar ATPase to phagosomes containing Leishmania donovani [164, 165], and Syt VII is needed for lysosomal membrane delivery [166]. The C2B domain of Syts has been demonstrated to bind to PI(3,4,5)P 3 ; in the presence of Ca 2 this specificity changes, and PI(4,5)P 2 is preferentially bound [167, 168]. Although much of the work currently examining Syt phosphoinositide binding has been done in vitro, a study examining phosphoinositide binding of the C2A domain of Syt I found that this probe colocalized with PI(4,5)P 2 -rich areas of the plasma membrane and TGN [169]. It is likely that future studies examining the intracellular localization of Syts will determine the extent of phosphoinositide interactions and whether these proteins contribute to phagosome maturation. This may be particularly relevant to SNARE activation for membrane fusion, as Syts coordinate the SNARE complexes that are needed to bring about membrane fusion during exocytosis (for a review, see ref. [170]). The mechanics of SNARE-mediated membrane fusion during phagosome maturation are likely to be highly similar to that which occurs during exocytosis. In both cases, SNAREs mediate fusion of vesicles with the Volume 92, September 2012 Journal of Leukocyte Biology 561

10 TABLE 3. Classes of PI3Ks and Their Roles in Phagosome Maturation Class Class IA Catalytic subunits: p110, p110, p110 Regulatory subunits: p85 and -, p50, p55 and - Class II C2, -, and - Class III Heterodimer: hvps34 with p150/vps15 In phagocytosis deactivates Rho GTPases Cdc42 and ARF6, activates Rac2 and ARF1 Generates PI(3,4,5)P 3 on phagosome membrane, which recruits Akt Generates PI(3,4,5)P 3, which recruits 4- and 5-inositol phosphatases? Downstream effects Allows pseudopod extension and phagocytic cup closure [44, 46] Akt deactivates Rab14 GAP AS160, resulting in Rab14 activity and phagosome interactions with early endosomes [150] Akt activation results in increased Rab7 expression during SP-A phagosome maturation [15] Breakdown of PI(3,4,5)P 3 to form PI3P, which promotes phagosome maturation [52]? p85 subunit acts as a GAP for Rab5? Results in Rab5 deactivation [123] C. elegans homolog PIKI-1 forms PI3P on May be responsible for initial phagosomal phagosomes PI3P, with Vps34 forming PI3P later in maturation [57] Generates PI3P PI3P recruits EEA-1 [126, 128], may play a role in Rab7 recruitment PI3P needed for Vamp7 SNARE recruitment [141, 142] Ca 2 release stimulates calmodulin/hvps34 interaction [163] TLR stimulation increases Vps34 membrane association and PI3P levels [17, 19] SLAMF1 recruits Vps34/Beclin1 to phagosome, leading to increased PI3P levels Activation of Syntaxin 6 and delivery of lysosomal components from TGN [158] Increased LC3-II levels indicating autophagy induction, increased phagosomal acidification [17] Autophagy induction [20] plasma membrane (the phagosome membrane having originated from the plasma membrane). Thus, it is likely that Syts may contribute to SNARE activation during phagosome maturation. CONCLUDING REMARKS PI3K activity has been shown to regulate multiple aspects of phagosome maturation (summarized in Table 3). Once thought to rely solely on PI3P formed by the class III enzyme hvps34, recent developments, such as the discovery of Akt on phagosomes and endosomes [56, 152], and the contributions of Rabs other than Rab5 and Rab7 [143, 150, 151], now suggest that phagosome maturation is affected by other PI3K isoforms as well. In addition, exciting developments in the field of TLR and SLAM signaling have indicated a role for these receptors in enhancing autophagosome maturation via PI3K activity [17 20]. These studies have highlighted commonalities shared between phagosome maturation and autophagy and the key role that PI3Ks play in both processes. More than 100 years after Ilya Metchnikoff first postulated that phagocytes have the ability to digest prey [171], there is still much to learn about this fascinating process. AUTHORSHIP E.P.T. wrote the manuscript. N.E.R. provided critical feedback and revised the manuscript. ACKNOWLEDGMENTS Funding was provided by Canadian Institutes of Health Research grant MOP to N.E.R. and a Canada Graduate Scholarship Doctoral Award (CGD-87892) to E.P.T. Sincere thanks go to the members of the Reiner Laboratory for insightful discussions and suggestions. We apologize to colleagues whose work could not be cited here due to space limitations. REFERENCES 1. Clemens, D. L., Horwitz,M. A. (1995) Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181, de Chastellier, C., Lang, T., Thilo, L. (1995) Phagocytic processing of the macrophage endoparasite, Mycobacterium avium, in comparison to phagosomes which contain Bacillus subtilis or latex beads. Eur. J. Cell Biol. 68, Suire, S., Coadwell, J., Ferguson, G. J., Davidson, K., Hawkins, P., Stephens, L. (2005) p84, a new G -activated regulatory subunit of the type IB phosphoinositide 3-kinase p110. Curr. Biol. 15, Voigt, P., Brock, C., Nurnberg, B., Schaefer, M. (2005) Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase. J. Biol. Chem. 280, Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, B., Zerial, M. (1992) The small GTPase Rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70, Gorvel, J. P., Chavrier, P., Zerial, M., Gruenberg, J. (1991) Rab5 controls early endosome fusion in vitro. Cell 64, Singer-Kruger, B., Stenmark, H., Dusterhoft, A., Philippsen, P., Yoo, J. S., Gallwitz, D., Zerial, M. (1994) Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J. Cell Biol. 125, Feng, Y., Press, B., Wandinger-Ness, A. (1995) Rab 7: an important regulator of late endocytic membrane traffic. J. Cell Biol. 131, Journal of Leukocyte Biology Volume 92, September