Thomas Weimbs

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.

Syntaxin 2 and Endobrevin Are Required for the Terminal Step of Cytokinesis in Mammalian Cells

The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.

Polarity Proteins Control Ciliogenesis via Kinesin Motor Interactions

BACKGROUNDnCilia are specialized organelles that play a fundamental role in several mammalian processes including left-right axis determination, sperm motility, and photoreceptor maintenance. Mutations in cilia-localized proteins have been linked to human diseases including cystic kidney disease and retinitis pigmentosa. Retinitis pigmentosa can be caused by loss-of-function mutations in the polarity protein Crumbs1 (CRB1), but the exact role of CRB1 in retinal function is unclear.nnnRESULTSnHere we show that CRB3, a CRB1-related protein found in epithelia, is localized to cilia and required for proper cilia formation. We also find that the Crumbs-associated Par3/Par6/aPKC polarity cassette localizes to cilia and regulates ciliogenesis. In addition, there appears to be an important role for the polarity-regulating 14-3-3 proteins in this process. Finally, we can demonstrate association of these polarity proteins with microtubules and the microtubular motor KIF3/Kinesin-II.nnnCONCLUSIONSnOur findings point to a heretofore unappreciated role for polarity proteins in cilia formation and provide a potentially unique insight into the pathogenesis of human kidney and retinal disease.

A model for structural similarity between different SNARE complexes based on sequence relationships

In the June 1998 issue of trends in CELL BIOLOGY, Götte and Fischer von Mollard summarized recent results in an updated picture of the structure and function of the SNARE machinery that mediates most if not all cellular membranefusion events (see Ref. 1 and references therein). The structure of the synaptic SNARE machinery has been studied in most detail, and it is clear now that the core structure involves two SNARE proteins at the target membrane (the t-SNAREs syntaxin 1 and SNAP-25) and one SNARE at the vesicle membrane (the v-SNARE synaptobrevin/VAMP). These three proteins form a stable trimeric complex held together by coiled-coil interactions between two domains of SNAP-25 and one each of syntaxin and synaptobrevin/VAMP. However, the structure of SNARE complexes at organelles other than the plasma membrane has remained more of a mystery. No SNAP-25-like proteins have been identified in those complexes, and, instead, a multitude of small v-SNARE-like proteins interact with syntaxin homologues on intracellular organelles. In some cases, evidence suggests that more than one v-SNARE-like protein is present in one complex1. We would like to add to this discussion our recent finding that vand t-SNAREs are evolutionarily related to each other, and suggest a hypothesis to explain the absence of SNAP-25 homologues and the presence of more than one v-SNARE in intracellular SNARE complexes. There are several homologues of vand t-SNAREs involved in fusion to different membrane compartments in the cell. It has been difficult to analyse sequence relationships between SNAREs because the only conserved regions show a propensity for heptad repeats, which occur in many unrelated proteins. Using profilebased sequence analysis, we demonstrated recently the evolutionary relationship of t-SNAREs of the syntaxin and SNAP-25 families2. Syntaxins contain one copy of a conserved ‘t-SNARE domain’ of 60 amino acids, whereas SNAP-25 proteins have two copies. These domains are identical to the coiled-coil domains that mediate interactions between SNARE proteins. A classification of the small v-SNARE-like proteins has been more difficult despite the fact that they share common features. We have now extended our sequence analysis of SNARE proteins using generalized profiles, which are derived from multiple alignments and contain information on which part of the sequence is most highly conserved and which regions of the sequence are likely to tolerate deletions or insertions3. Iteratively refined profile searches are a sensitive method of detecting distant sequence similarities, and, unlike protein threading methods, similarities found by profile searches typically reflect relationships based on divergent rather than convergent evolution. We created a profile using the sequence of the Golgi v-SNARE Bos1p and recently reported sequences of related proteins. Database searches with this profile revealed several new and uncharacterized, yet clearly related, yeast and nematode sequences, which were included into the profile. The resulting refined profile, consisting of the central conserved regions and the C-terminal membrane anchor, was used for further database searches. Surprisingly, the highest-scoring matches were members of the syntaxin and synaptobrevin/VAMP families, the best matching of which indicated a statistically significant relationship. This strongly suggests that the Bos1-, syntaxinand synaptobrevin/VAMPfamilies are evolutionarily related to each other. Since we had previously already reported the relationship between the syntaxin and SNAP-25 families and Bet1p2, we conclude that all currently known vand t-SNAREs are likely to have evolved from a single common ancestral gene and belong to a common superfamily. Five distinct SNARE subfamilies can be identified by nearestneighbour dendrogram analysis (see Box 1). Figure 1 shows an alignment of the homologous sequences of representative members of these subfamilies. Several features are striking: the conservation of the heptad repeats, a cluster of basic amino acids that separates the coiled-coil and transmembrane domains, and the conservation of a glutamine residue in the d-position of a heptad repeat in all but the synaptobrevin/VAMP family. The evolutionary relationship of SNARE proteins leads us to propose the following hypothesis. There is reason to believe that members of the SNAP-25 family are exclusively involved in fusion at the plasma membrane. First, the completely sequenced genome of Saccharomyces cerevisiae contains only two SNAP-25 members2, Sec9p and Spo20p (YMR017w), both of which function in fusion to the plasma membrane4. Second, in mammalian systems, only two family members have been characterized: SNAP-25 at the presynaptic plasma membrane and SNAP-23 at plasma membranes in other tissues5. Given the evolutionary relationship between the coiledcoil domains of all SNAREs, we suggest that the intracellular SNARE complexes are structurally similar to the synaptic SNARE complex in that the number of coiled-coil domains in each complex is fixed. One molecule of SNAP-25, which contributes two coiled-coil domains in the synaptic complex, could be substituted by two small SNAREs of the synaptobrevin/VAMP-, Bet1and/or Bos1-families, which could therefore be regarded as ‘half-SNAP-25s’. Several arrangements are possible, one of which is depicted in Figure 2b. It is possible that the binding of one or two given small SNARE(s) to a syntaxin isoform could alter the binding properties of the resulting dimer or trimer, thereby creating t-SNAREs with different specificities – which could explain how one single BOX 1 – FURTHER INFORMATION

Apical targeting in polarized epithelial cells: There's more afloat than rafts

Most metazoan cells are polarized. A crucial aspect of this polarization is that the plasma membrane is divided into two or more domains with different protein and lipid compositions or example, the apical and basolateral domains of epithelial cells or the axonal and somatodendritic domains of neurons. This polarity is established and maintained by highly specific vesicular membrane transport in the biosynthetic, endocytic and transcytotic pathways. Two important concepts, the SNARE and the raft hypotheses, have been developed that together promise at least a partial understanding of the underlying general mechanisms that ensure the necessary specificity of these pathways.

Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells

We have examined the role of rab3b in epithelial cells. In MDCK cells, rab3b localizes to vesicular structures containing the polymeric immunoglobulin receptor (pIgR) and located subjacent to the apical surface. We found that GTP-bound rab3b directly interacts with the cytoplasmic domain of pIgR. Binding of dIgA to pIgR causes a dissociation of the interaction with rab3b, a process that requires dIgA-mediated signaling, Arg657 in the cytoplasmic domain of pIgR, and possibly GTP hydrolysis by rab3b. Binding of dIgA to pIgR at the basolateral surface stimulates subsequent transcytosis to the apical surface. Overexpression of GTP-locked rab3b inhibits dIgA-stimulated transcytosis. Together, our data demonstrate that a rab protein can bind directly to a specific cargo protein and thereby control its trafficking.

Apical targeting of syntaxin 3 is essential for epithelial cell polarity

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

Polycystin-1 regulates STAT activity by a dual mechanism

Mutations in polycystin-1 (PC1) lead to autosomal-dominant polycystic kidney disease (ADPKD), a leading cause of renal failure for which no treatment is available. PC1 is an integral membrane protein, which has been implicated in the regulation of multiple signaling pathways including the JAK/STAT pathway. Here we show that membrane-anchored PC1 activates STAT3 in a JAK2-dependent manner, leading to tyrosine phosphorylation and transcriptional activity. The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation. Tail-cleavage abolishes the ability of PC1 to directly activate STAT3 but the cleaved PC1 tail now coactivates STAT3 in a mechanism requiring STAT phosphorylation by cytokines or growth factors. This leads to an exaggerated cytokine response. Hence, PC1 can regulate STAT activity by a dual mechanism. In ADPKD kidneys PC1 tail fragments are overexpressed, including a unique ∼15-kDa fragment (P15). STAT3 is strongly activated in cyst-lining epithelial cells in human ADPKD, and orthologous and nonorthologous polycystic mouse models. STAT3 is also activated in developing, postnatal kidneys but inactivated in adult kidneys. These results indicate that STAT3 signaling is regulated by PC1 and is a driving factor for renal epithelial proliferation during normal renal development and during cyst growth.

Prospects for mTOR Inhibitor Use in Patients with Polycystic Kidney Disease and Hamartomatous Diseases

Mammalian target of rapamycin (mTOR) is the core component of two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and analogues. mTORC2 is impeded only in some cell types by prolonged exposure to these compounds. mTOR activation is linked to tubular cell proliferation in animal models and human autosomal dominant polycystic kidney disease (ADPKD). mTOR inhibitors impede cell proliferation and cyst growth in polycystic kidney disease (PKD) models. After renal transplantation, two small retrospective studies suggested that mTOR was more effective than calcineurin inhibitor-based immunosuppression in limiting kidney and/or liver enlargement. By inhibiting vascular remodeling, angiogenesis, and fibrogenesis, mTOR inhibitors may attenuate nephroangiosclerosis, cyst growth, and interstitial fibrosis. Thus, they may benefit ADPKD at multiple levels. However, mTOR inhibition is not without risks and side effects, mostly dose-dependent. Under certain conditions, mTOR inhibition interferes with adaptive increases in renal proliferation necessary for recovery from injury. They restrict Akt activation, nitric oxide synthesis, and endothelial cell survival (downstream from mTORC2) and potentially increase the risk for glomerular and peritubular capillary loss, vasospasm, and hypertension. They impair podocyte integrity pathways and may predispose to glomerular injury. Administration of mTOR inhibitors is discontinued because of side effects in up to 40% of transplant recipients. Currently, treatment with mTOR inhibitors should not be recommended to treat ADPKD. Results of ongoing studies must be awaited and patients informed accordingly. If effective, lower dosages than those used to prevent rejection would minimize side effects. Combination therapy with other effective drugs could improve tolerability and results.

Folate-Conjugated Rapamycin Slows Progression of Polycystic Kidney Disease

Activation of the mammalian target of rapamycin (mTOR) signaling pathway is aberrant in autosomal-dominant polycystic kidney disease (ADPKD). The mTOR inhibitors, such as rapamycin, ameliorate PKD in rodent models, but clinical trials have not shown benefit, possibly as a result of low tissue concentrations of rapamycin at clinically tolerable doses. To overcome this limitation, we synthesized a folate-conjugated form of rapamycin (FC-rapa) that is taken up by folate receptor-mediated endocytosis and cleaved intracellularly to reconstitute the active drug. We found that renal cyst-lining cells highly express the folate receptor in ADPKD and mouse models. In vitro, FC-rapa inhibited mTOR activity in a dose- and folate receptor-dependent manner. Treatment of a PKD mouse model with FC-rapa inhibited mTOR in the target tissue, strongly attenuated proliferation and growth of renal cysts and preserved renal function. Furthermore, FC-rapa inhibited mTOR activity in the kidney but not in other organs. In summary, these results suggest that targeting the kidney using FC-rapa may overcome the significant side effects and lack of renal efficacy observed in clinical trials with mTOR inhibitors in ADPKD.