Apical Recycling Research Articles

Insights into acidosis-induced regulation of SLC26A4 (pendrin) and SLC4A9 (AE4) transporters using three-dimensional morphometric analysis of β-intercalated cells

The purpose of this study was to examine the three-dimensional (3-D) expression and distribution of anion transporters pendrin (SLC26A4) and anion exchanger (AE)4 (SLC4A9) in β-intercalated cells (β-ICs) of the rabbit cortical collecting duct (CCD) to better characterize the adaptation to acid-base disturbances. Confocal analysis and 3-D reconstruction of β-ICs, using identifiers of the nucleus and zona occludens, permitted the specific orientation of cells from normal, acidotic, and recovering rabbits, so that adaptive changes could be quantified and compared. The pendrin cap likely mediates apical Cl(-)/HCO3 (-) exchange, but it was also found beneath the zona occludens and in early endosomes, some of which may recycle back to the apical membrane via Rab11a(+) vesicles. Acidosis reduced the size of the pendrin cap, observed as a large decrease in cap volume above and below the zona occludens, and the volume of the Rab11a(+) apical recycling compartment. Correction of the acidosis over 12-18 h reversed these changes. Consistent with its proposed function in the basolateral exit of Na(+) via Na(+)-HCO3 (-) cotransport, AE4 was expressed as a barrel-like structure in the lateral membrane of β-ICs. Acidosis reduced AE4 expression in β-ICs, but this was rapidly reversed during the recovery from acidosis. The coordinate regulation of pendrin and AE4 during acidosis and recovery is likely to affect the magnitude of acid-base and possibly Na(+) transport across the CCD. In conclusion, acidosis induces a downregulation of AE expression in β-ICs and a diminished presence of pendrin in apical recycling endosomes.

Clathrin and its adaptor AP1 are required for apical sorting of GPI‐anchored proteins in biosynthetic and recycling routes in MDCK cells (598.4)

To fulfill their functions, epithelial cells establish asymmetric protein distributions in apical and basolateral plasma membranes. To date, the role of clathrin and its adaptor, the AP1 complex, has been documented exclusively for basolateral protein trafficking in mammalian epithelial cells. We found that the apical localization of endogenous glycosylphosphatidylinositol (GPI)‐anchored proteins is severely affected by gene silencing of the clathrin heavy chain and components of the AP1 adaptor complex in MDCK cells. In these cells, apical targeting is affected not only for GPI‐ anchored proteins but also for endogenous secretory proteins and the apical‐destined t‐SNARE, syntaxin 3. Analysis of apical secretion of lysoGPI‐APs reveals that the biosynthetic delivery of GPI‐APs toward apical plasma membrane is impaired in these cells. Furthermore, apical recycling and transcytosis from basolateral to apical membranes of GPI‐APs are also impaired in these cells. Our results formally demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.Grant Funding Source: Swiss National Sciences Foundation Assistant Professorship

Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

Drebrin E depletion in human intestinal epithelial cells mimics Rab8a loss of function

Intestinal epithelial cells are highly polarized and exhibit a complex architecture with a columnar shape and a specialized apical surface supporting microvilli organized in a brush border. These microvilli are rooted in a dense meshwork of acto-myosin called the terminal web. We have shown recently that Drebrin E, an F-actin-binding protein, is a key protein for the organization of the terminal web and the brush border. Drebrin E is also required for the columnar cell shape of Caco2 cells (human colonic cells). Here, we found that the subcellular localization of several apical markers including dipeptidyl peptidase IV (DPPIV) was strikingly modified in Drebrin E-depleted Caco2 cells. Instead of being mostly present at the apical surface, these proteins are accumulated in an enlarged subapical compartment. Using known intracellular markers, we show by both confocal and electron microscopy that this compartment is related to lysosomes. We also demonstrate that the enrichment of DPPIV in this compartment originates from apical endocytosis and that depletion of Rab8a induces an accumulation of apical proteins in a similar compartment. Consistent with this, the phenotype observed in Drebrin E knock-down Caco2 cells shares some features with a pathology called microvillar inclusion disease (MVID) involving both Myosin Vb and Rab8a. Taken together, these results suggest that Drebrin E redirects the apical recycling pathway in intestinal epithelial cells to the lysosomes, demonstrating that Drebrin E is a key regulator in apical trafficking in Caco2 cells.

Cdc42 GTPase dynamics control directional growth responses.

Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca(2+) influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca(2+) influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca(2+). Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca(2+). Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca(2+). The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca(2+) influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca(2+)-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

Class III Phosphoinositide 3‐Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling

Recycling is a limiting step for receptor-mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan-class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP-2, causing surface down-regulation. GFP-(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002-treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin-GFP and dependence of dynamin-GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K-III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

Retromer maintains basolateral distribution of the type II TGF-β receptor via the recycling endosome

Transforming growth factor β (TGF-β) is critical for the development and maintenance of epithelial structures. Because receptor localization and trafficking affect the cellular and organismal response to TGF-β, the present study was designed to address how such homeostatic control is regulated. To that end, we identify a new role for the mammalian retromer complex in maintaining basolateral plasma membrane expression of the type II TGF-β receptor (TβRII). Retromer and TβRII associate in the presence or absence of TGF-β ligand. After retromer knockdown, although TβRII internalization and trafficking to a Rab5-positive compartment occur as in wild-type cells, receptor recycling is inhibited. This results in TβRII mislocalization from the basolateral to both the basolateral and apical plasma membranes independent of Golgi transit and the Rab11-positive apical recycling endosome. The data support a model in which, after initial basolateral TβRII delivery, steady-state polarized TβRII expression is maintained by retromer/TβRII binding and delivery to the common recycling endosome.

176 Loss of Functional MYO5B in CaCo2-BBe Cells and Navajo MVID Patient Enterocytes Leads to Loss of Polarity and Deficits in Maintenance of Microvilli

Myosin Vb (MYO5B) is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains in polarized epithelial cells. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that leads to the loss of microvilli in intestinal enterocytes and chronic unremitting diarrhea in the affected newborns. Understanding how mutations in MYO5B lead to aberrant enterocyte phenotype will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking, microvilli formation, and MVID. We hypothesize that MYO5B plays a crucial role in apical trafficking and polarization in enterocytes, and that defective MYO5B in MVID patients mislocalizes Rab small GTPases leading to disruption in apical transport and loss of polarity in enterocytes. To test this hypothesis we have established cellular models of aberrant enterocyte apical trafficking by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. MYO5B KD knockdown (KD) caused a loss of microvilli and dispersal of Rab8a and Rab11acontaining vesicles from their usual subapical distribution to diffusely cytoplasmic in the cells. MYO5B KD elicited an increase in claudin-2 and a decrease in claudin-1 expression as well as a decrease in lateral membrane staining for p120-catenin (p120). MYO5B KD also caused a loss of apical active GTP-bound cdc42 in the cells. Rescue of theMYO5B KDwith synthetic MYO5B wild type elicited recovery of normal microvilli. However, re-expression of MYO5B(P660L), the Navajo MVID mutation, failed to rescue and in addition caused the formation of microvillus inclusions. Navajo MVID MYO5B(P660L) patient samples demonstrated a loss of p120 and a pseudostratified columnar epithelium at the villus tips where ezrin-staining microvillus inclusions were most predominant. Patient biopsies also showed aberrant localization of MYO5B, Rab8a, Rab11a, Rab11-FIP2, DPPIV, and Ezrin. Rab11a and MYO5B localized around microvillus inclusions, while Rab8a staining was diffuse. Importantly, while a severe loss of brush border was observed in cells at the tips of microvilli, the cells in the mid-villus appeared to have a preserved normal brush border. Our results suggest that MYO5B regulates the polarity of intestinal epithelial cells, thereby maintaining the functional brush border. The normal brush border seen in the proximal villi in the Navajo MVID patients suggests that the pathogenesis of MVID lies in a loss of polarity in enterocytes at the tips of intestinal villi.

Role of Rab11a Positive Compartments in Endocytic Traffic in Primary Proximal Tubule Cells

The proximal tubule (PT) reabsorbs ~ 70% of sodium, bicarbonate, chloride ions, phosphate, glucose, water, and the majority of the plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell function, the organization of the endocytic pathway in these cells remains poorly understood. We have used live‐cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PT cells isolated from mice. Cells from the S1 segment could be distinguished from more distal segments by their robust uptake of albumin and low expression of γ‐glutamyltranspeptidase. Rab11a in these cells is localized to numerous dynamic spherical compartments that resemble the apical vacuoles found in electron microscopy analysis of cultured PT cells. These structures are highly dynamic and receive both membrane and fluid phase cargo. In contrast, Rab11a in immortalized kidney cell lines is localized to a condensed and largely tubular apical recycling compartment through which recycling membrane proteins (but not fluid phase cargoes) transit. The unusual morphology of Rab11a‐positive compartments in primary PT cells may reflect the demands of handling a high endocytic load and suggests a possible role for Rab11a in early sorting steps of fluid and membrane proteins in these cells. This research was supported by NIH R01 DK54407 and P30 DK079307.

Keratin K18 Increases Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Surface Expression by Binding to Its C-terminal Hydrophobic Patch

CFTR function is tightly regulated by many interacting proteins. Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. K18 controls the function of CFTR. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.

The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na+ transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,β,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.

Actin polymerization controls the activation of multidrug efflux at fertilization by translocation and fine-scale positioning of ABCB1 on microvilli

Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin polymerization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.

MYO5B Mutations in Patients With Microvillus Inclusion Disease Presenting With Transient Renal Fanconi Syndrome

: Microvillus inclusion disease (MVID) is a rare congenital enteropathy associated with brush border atrophy and reduced expression of enzymes at the enterocytes' apical surface. MVID is associated with mutations in the MYO5B gene, which is expressed in all epithelial tissues. Whether organs other than the intestine are affected in MVID is unclear. We report 2 patients with MVID that developed renal Fanconi syndrome while receiving total parenteral nutrition. Renal Fanconi syndrome has been correlated to apical plasma membrane defects in kidney proximal tubular epithelial cells. The aim of the present study was to determine whether MYO5B mutations in these patients correlate with similar apical plasma membrane defects in renal tubular epithelial cells as observed in the intestine. : Biopsies from kidney, duodenum, ileum, jejunum, and colon of 2 patients with MVID carrying MYO5B mutations and of age-matched controls were fixed in paraffin and analyzed with immunohistochemistry and transmission electron microscopy. : Structural defects of the brush border and apical recycling endosome organization are observed in enterocytes of all of the segments of the small intestine and colon. MYO5B mutations in patients with MVID with renal Fanconi syndrome do not correlate with aberrant apical plasma membrane morphology or altered apical recycling endosome organization in renal tubular epithelial cells. : MYO5B mutations have divergent effects on the apical membrane system in kidney and intestinal epithelial cells. Epithelial defects presented in MVID are therefore likely triggered by intestine-specific factors, the identification of which may provide new targets and open avenues for the development of alternative therapeutic strategies to combat this devastating disease.

Analysis of Polarized Membrane Traffic in Hepatocytes and Hepatic Cell Lines

The protocols described in this unit were developed to monitor membrane traffic in cultured cell monolayers that display hepatic polarity. In general, the assays are designed to visualize and/or quantitate membrane trafficking by monitoring the fates of antibodies bound to specific membrane proteins. We first describe how to infect cells with recombinant adenovirus, the preferred method for introducing exogenous genes into hepatic cells. We next provide a morphological assay to monitor basolateral to apical transcytosis. In a supporting protocol, we describe how to visualize apical recycling and/or retention. In an additional supporting protocol, we provide a semi-quantitative method to measure the relative extents of apical delivery. Finally, we describe quantitative assays to measure basolateral internalization and recycling. The methods presented in this unit provide a relatively simple, yet powerful approach to examining hepatic membrane traffic.

Rab11 Is a Useful Tool for the Diagnosis of Microvillous Inclusion Disease

Microvillous inclusion disease (MVID) is a congenital condition presenting with intractable diarrhea. Biopsies demonstrate abnormal apical PAS and CD10 staining in surface enterocytes correlating with the presence of characteristic cytoplasmic inclusions. MVID has been linked to mutations in myosin Vb, important in apical membrane recycling. Rab11 associates with myosin Vb in vesicle membranes and is also integral in recycling plasma membrane components. The authors performed Rab11 immunostaining on biopsies from 7 MVID cases, 10 normal small intestines, and 10 with chronic enteritis. In MVID cases, Rab11 showed diffuse apical cytoplasmic staining of surface enterocytes in a pattern similar to PAS and CD10, which was absent in all the 20 control cases. Ultrastructural examination confirmed localization to the external surface of MVID cytoplasmic inclusions. Rab11 staining may be a useful adjunct in MVID diagnosis and the results support that myosin Vb dysfunction is important in the pathogenesis of MVID.

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.

Functional analysis of the putative AAA ATPase AipA localizing at the endocytic sites in the filamentous fungus Aspergillus oryzae

We searched for novel components involved in Aspergillus oryzae endocytosis by yeast two-hybrid (YTH) screening. Using the endocytic marker protein AoAbp1 (A. oryzae homolog of Saccharomyces cerevisiae Abp1p) as bait, a putative AAA (ATPases associated with diverse cellular activities) ATPase encoded by a gene termed aipA (AoAbp1 interacting protein) was identified. Further YTH analyses showed that the 346-370 amino-acid region of AipA interacts with the SH3 (Src homology 3) domains of AoAbp1. Moreover, AipA colocalized with AoAbp1 at the tip region, suggesting that AipA functions in endocytosis. Although aipA disruptants did not display defective growth, an aipA-overexpressing strain displayed impaired growth and wider hyphal morphology. In addition, we generated strains that overexpressed either aipA(K542A) or aipA(E596Q) , whose mutations were introduced into the AAA ATPase domain of AipA and would cause the defect of ATPase activity. In contrast to the aipA-overexpressing strain, neither aipA(K542A) - nor aipA(E596Q) -overexpressing strains showed defective growth. Moreover, only the aipA-overexpressing strain displayed a defect of FM4-64 transport to the Spitzenkörper, suggesting that AipA negatively regulates apical endocytic recycling and that the AAA ATPase domain of AipA is crucial for its function.

Rab11-FIP2 influences multiple components of the endosomal system in polarized MDCK cells.

The Rab11 Family Interacting Proteins (Rab11-FIPs) are hypothesized to regulate sequential steps in the apical recycling and transcytotic pathways of polarized epithelial cells. Previous studies have suggested that Rab11-FIP proteins assemble into multi-protein complexes regulating plasma membrane recycling. Rab11-FIP2 interacts with both myosin Vb and Rab11. Recent investigations have noted that that Rab11-FIP2 mutants [Rab11-FIP2(129-512), also designated Rab11-FIP2(ΔC2) and Rab11-FIP2(S229A, R413G), also designated Rab11-FIP2(SARG)], are potent inhibitors of transcytosis in polarized MDCK cells. Interestingly, Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), also altered the morphology of the EEA-1 positive early endosomal compartment. These findings suggested that Rab11-FIP2 mutants could differentiate different points along the recycling pathway. We therefore sought to investigate whether Rab11-FIP2 is a general regulator of the early endosomal system. Both Rab11-FIP2 mutants altered the localization and co-localized with dynein heavy chain. In contrast, both clathrin heavy chain and AP-1 accumulated with membranes containing Rab11-FIP2(SARG), but not with Rab11-FIP2(ΔC2). Expression of Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), caused clustering of early endosomal markers Rab5b, Epsin 4 and IQGAP1, around a collapsed Rab11-FIP2 containing membranous cisternum. Interestingly, neither Rab11-FIP2 mutant had any effect on the distribution of Rab5a, a classical early endosome marker. The results support the view that Rab11-FIP2 may influence microtubule-dependent centripetal movement of subsets of early endosomes as well as processing through the common and recycling endosomal systems.

A role for endocytic recycling in hyphal growth

Actin plays multiple complex roles in cell growth and cell shape. Recently it was demonstrated that actin patches, which represent sites of endocytosis, are present in a sub-apical collar at growing tips of hyphae and germ tubes of filamentous fungi. It is now clear that this zone of endocytosis is necessary for filamentous growth to proceed. In this review evidence for the role of these endocytic sites in hyphal growth is examined. One possibility if that the role of the sub-apical collar is associated with endocytic recycling of polarized material at the hyphal tip. The ‘Apical Recycling Model’ accounts for this role and predicts the need for a balance between endocytosis and exocytosis at the hyphal tip to control growth and cell shape. Other cell differentiation events, including appressorium formation and Aspergillus conidiophore development may also be explained by this model.