Jeffrey J. Talbot, Xuewen Song, Xiaofang Wang, Markus M. Rinschen, Nicholas Doerr, Wells B. LaRiviere, Bernhard Schermer,§ York P. Pei, Vicente E. Torres, and Thomas Weimbs (2014)
The Cleaved Cytoplasmic Tail of Polycystin-1 Regulates Src-Dependent STAT3 Activation
J Am Soc Nephrol 25: Published online before print February 27, 2014

Abstract: Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we show that PC1-p30 interacts with the nonreceptor tyrosine kinase Src, resulting in Src- dependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30–mediated activation of Src/ STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cyto- kine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1- p30, cAMP dampened EGFR- or IL-6–dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) rat model, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP, resulting in Src-dependent activation of STAT3 and a proliferative response.

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Thomas Weimbs, Jeffrey J. Talbot (2013)
STAT3 signaling in polycystic kidney disease,
Drug Discovery Today: Disease Mechanisms (in press) Available online 26 March 2013

Abstract: Mutations in the gene coding for the integral membrane protein polycystin-1 (PC1) are the cause of most cases of autosomal-dominant polycystic kidney disease (ADPKD), a very common disease that leads to kidney failure and currently lacks approved treatment. Recent work has revealed that PC1 can regulate the transcription factor STAT3, and that STAT3 is aberrantly activated in the kidneys of ADPKD patients and PKD mouse models. Recent approaches to directly inhibit STAT3 in PKD mouse models have been promising. Numerous signaling pathways are known to activate STAT3 and many have long been implicated in the pathogenesis of PKD – such as EGF/EGFR, HGF/c-Met, Src. However, a role of STAT3 in the pathogenesis of PKD had never been considered until now. Here, we review the current findings that suggest that STAT3 is a promising target for the treatment of PKD.

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Weimbs, T., Olsan, E. E., & Talbot, J. J. (2013)
Regulation of STATs by polycystin-1 and their role in polycystic kidney disease.
JAK-STAT, 2(2), e23650-1 - e23650-9

Abstract: Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disease caused by mutations in the gene coding for polycystin-1 (PC1). PC1 can regulate STAT transcription factors by a novel, dual mechanism. STAT3 and STAT6 are aberrantly activated in renal cysts. Genetic and pharmacological approaches to inhibit STAT3 or STAT6 have led to promising results in ADPKD mouse models. Here, we review current findings that lead to a model of PC1 as a key regulator of STAT signaling in renal tubule cells. we discuss how PC1 may orchestrate appropriate epithelial responses to renal injury, and how this system may lead to aberrant STAT activation in ADPKD thereby causing inappropriate activation of tissue repair programs that culminate in renal cyst growth and fibrosis.

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Jonathan M. Shillingford, Christopher P. Leamon, Iontcho R. Vlahov, and Thomas Weimbs (2012)
Folate-Conjugated Rapamycin Slows Progression of Polycystic Kidney Disease
J Am Soc Nephrol 23, 1674–1681

Abstract: 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 in- tracellularly 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. Further- more, 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.

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Stayner C, Shields J, Slobbe L, Shillingford JM, Weimbs T, Eccles MR (2012)
Rapamycin-mediated suppression of renal cyst expansion in del34 Pkd1(-/-) mutant mouse embryos; an investigation of the feasibility of renal cyst prevention in the fetus.
Nephrology 17, 739–747

Abstract: Stayner et al. investigated the effect of rapamycin on polycystic kidney disease in utero. Their data showed the potential beneficial effects of rapamycin administered during pregnancy in a rodent model of PKD, while cyst number remains unchanged; the size of the cysts is reduced by rapamycin treatment. Furthermore, they also showed that the inhibition of the mTOR pathway by rapamycin most likely are not mediated via pax2 in their experimental animal model. The result provides evidence of rapamycin may be therapeutically useful for slowing the progression of PKD with a prenatal onset. ABSTRACT: Aim:  Polycystic kidney disease (PKD) in humans encompasses a group of disorders featuring kidney cyst expansion within the first decade (recessive PKD) or beyond the fourth to fifth decade of life (dominant PKD). Autosomal dominant PKD (ADPKD) is caused by mutations in PKD1 or PKD2 genes, and involves cyst formation beginning in utero. Like recessive PKD, ADPKD leads to end-stage kidney disease. Inhibition of mTOR signaling was recently found to halt cyst formation in adult ADPKD mice. In contrast, no studies have investigated potential treatments to prevent cyst formation in utero in recessive PKD. Given that homozygous Pkd1 mutant mice exhibit cyst formation in utero, we decided to investigate whether mTOR inhibition in utero ameliorates kidney cyst formation in fetal Pkd1homozygous mutant mice. Methods:  Pregnant Pkd1(+/-) female mice (mated with Pkd1(+/-) males) were treated with rapamycin from E14.5 to E17.5. Fetal kidneys were dissected, genotyped and evaluated by cyst size as well as expression of the developmental marker, Pax2. Results:  Numerous cysts were present in Pkd1(-/-) kidneys, which were twice the weight of wildtype kidneys. Cyst size was reduced by a third in rapamycin treated Pkd1(-/-) kidney sections and kidney mass was reduced to near wildtype levels. However, some lethality was observed in Pkd1(-/-) null embryos. Moreover, total cyst number was not reduced compared to control embryos. Pax2 expression and kidney development were unaltered in rapamycin treated mice. Conclusion:  Rapamycin treatment reduces cyst formation in utero in Pkd1(-/-) mutant mice, therefore the prevention of kidney cyst expansion in utero by mTOR inhibition is feasible. In contrast, selective rapamycin-associated lethality would likely limit its usefulness as a treatment in utero.

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Erin E. Olsan, Sambuddho Mukherjee, Beatrix Wulkersdorfer, Jonathan M. Shillingford, Adrian J. Giovannone, Gueorgui Todorov, Xuewen Song, York Pei, and Thomas Weimbs (2011)
Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease.
Proceedings of the National Academy of Sciences of the USA 108, 18067-18072

Abstract: Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.

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Elena Reales, Nikunj Sharma, Seng Hui Low, Heike Fölsch, Thomas Weimbs (2011)
Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity.
PLoS ONE 6(6): e21181

Abstract: Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.

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Talbot, J, J Shillingford, S Vasanth, N Doerr, S Mukherjee, M Kinter, T Watnick, and T Weimbs (2011)
Polycystin-1 Regulates STAT Activity by a Dual Mechanism.
Proceedings of the National Academy of Sciences of the USA 108, 7985-7990

Abstract: 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 phosphory- lation 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 over-expressed, 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.

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Weimbs T (2011)
Third-Hit Signaling in Renal Cyst Formation.
J. Am. Soc. Nephrol. 22, 793-795

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VE Torres, A Boletta, A Chapman, V Gattone, Y Pei, Q Qian, DP Wallace, T Weimbs, and RP Wuthrich (2010)
Prospects for mTOR Inhibitor Use in Patients with Polycystic Kidney Disease and Hamartomatous Diseases.
Clin. J. Am. Soc. Nephrol., 5(7):1312-29

Abstract: 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 remod- eling, 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 (down- stream 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.

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Shillingford, JM, Piontek, KB, Germino, GG and Weimbs, T (2010)
Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1.
J Am Soc Nephrol, 21, 489-97

Abstract: Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

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Abinash C. Mistry, Rickta Mallick, Janet D. Klein, Thomas Weimbs, Jeff M. Sands, and Otto Froehlich (2009)
Syntaxin specificity of aquaporins in the inner medullary collecting duct.
Am J Physiol Renal Physiol.
297, 292-300

Abstract: Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target-SNARE (t-SNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-MDCK cells subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pulldown studies, snapin detected AQP2, syntaxin-3, syntaxin-4 and SNAP23 from inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were co-injected with mRNAs of SNARE components (snapin + syntaxin-3 + SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin + syntaxin-4 + SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP/syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus, our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion.

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Weimbs T. (2007)
Polycystic kidney disease and renal injury repair: common pathways, fluid flow and the function of polycystin-1.
Am J Physiol Renal Physiol.
293, F1423–F1432

Abstract: The root cause for most cases of autosomal-dominant polycystic kidney disease (ADPKD) are mutations in the polycystin-1 (PC1) gene. While PC1 has been implicated in a perplexing variety of protein interactions and signaling pathways, it is unknown what its normal function is and why its disruption leads to proliferation of renal epithelial cells. Recent results suggest that PC1 is involved in mechanotransduction by primary cilia measuring the degree of luminal fluid flow. PC1 has also recently been shown to regulate the mTOR and STAT6 pathways. These two pathways are normally dormant in the healthy kidney but are activated in response to injury and appear to drive a proliferative repair response. This review is developing the idea that a critical function of polycystin-1 and primary cilia in the adult kidney may be to sense renal injury by detecting changes in luminal fluid flow and to trigger proliferation. Constitutive activation of these pathways in ADPKD would lead to the futile attempt to repair a non-existing injury, resulting in cyst growth. The existence of many known cellular and molecular similarities between renal repair and ADPKD supports this model.

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Shipan Dai, Ying Zhang, Thomas Weimbs, Michael B Yaffe, and Daoguo Zhou (2007)
Bacteria-generated PtdIns(3)P recruits VAMP8 to facilitate phagocytosis.
Traffic 8, 1365–1374

Abstract: Salmonella enterica serovar Typhimurium invades non-phagocytic cells by inducing macropinocytosis. SopB is involved in modulating actin dynamics to promote Salmonella-induced invasion. We report here that SopB-generated PtdIns(3)P binds VAMP8/endobrevin to promote efficient bacterial phagocytosis. VAMP8 is recruited to Salmonella-induced macropinosomes in a nocodazole-dependent, but Brefeldin A-independent, manner. We found that VAMP8 directly binds to and colocalizes with PtdIns(3)P. The inositol phosphatase activity of SopB is required for PtdIns(3)P and VAMP8 accumulation, while wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, has no effect. Knockdown of endogenous VAMP8 by small interfering RNA or expression of a truncated VAMP8 (1-79aa) reduces the invasion level of wild-type Salmonella to that of the phosphatase-deficient SopB(C460S) mutant. Our study demonstrates that Salmonella exploit host SNARE proteins and vesicle trafficking to promote bacterial entry.

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Christiansen JJ, Weimbs T, Bander N, and Rajasekaran AK (2006)
Differing effects of microtubule depolymerizing and stabilizing chemotherapeutic agents on t-SNARE–mediated apical targeting of prostate-specific membrane antigen.
Mol Cancer Ther 5, 2468–73

Abstract: Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.

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Weimbs, T. (2006)
Regulation of mTOR by Polycystin-1: is Polycystic Kidney Disease a Case of Futile Repair?
Cell Cycle 5, 2425-2429

Abstract: Recent work has uncovered a functional link between polycystin-1 (PC1), the protein affected in autosomal-dominant polycystic kidney disease (ADPKD) and tuberin, the protein affected in tuberous sclerosis complex (TSC). These data suggest that PC1 functions by inducing the formation of a complex with tuberin and the Ser/Thr kinase mTOR thereby inhibiting mTOR activity. In normal, adult kidney, mTOR is inactive. However, it is activated in response to insults and required for proliferative and hyperthrophic repair processes. We propose a model in which the PC1-tuberin-mTOR complex functions to sense renal insults, possibly by ciliary mechanotransduction, and regulates the activity of mTOR to trigger a formal repair program. In ADPKD, defects in PC1 would lead to constitutive activation of mTOR, and the affected cells would be engaged in a permanent state of futile repair leading to the formation and growth of renal cysts. The mTOR inhibitor rapamycin has proven highly effective in preventing and even reversing cyst growth in rodent models of polycystic kidney disease resulting in preservation of renal function. mTOR inhibitors, already in clinical use as immunosuppressants, may therefore be promising for future therapeutic approaches for ADPKD.

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Kidd GJ, Yadav VK, Huang P, Brand SL, Low SH, Weimbs T, Trapp BD (2006)
A dual tyrosine-leucine motif mediates myelin protein P(0) targeting in MDCK cells.
Glia 54, 135-145

Abstract: Differential targeting of myelin proteins to multiple, biochemically and functionally distinct Schwann cell plasma membrane domains is essential for myelin formation. In this study, we investigated whether the myelin protein P0 contains targeting signals using Madin-Darby canine kidney (MDCK) cells. By confocal microscopy, P0 was localized to MDCK cell basolateral membranes. C-terminal deletion resulted in apical accumulation, and stepwise deletions defined a 15-mer region that was required for basolateral targeting. Alanine substitutions within this region identified the YAML sequence as a functional tyrosine-based targeting signal, with the ML sequence serving as a secondary leucine-based signal. Replacement of the P0 ectodomain with green fluorescent protein altered the distribution of constructs lacking the YAML signal. Coexpression of the myelin-associated glycoprotein did not alter P0 distribution in MDCK cells. The results indicate that P0 contains a hierarchy of targeting signals, which may contribute to P0 localization in myelinating Schwann cells and the pathogenesis in human disease.

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Sharma N, Low SH, Misra S, Pallavi B, Weimbs T (2006)
Apical targeting of syntaxin 3 is essential for epithelial cell polarity
The Journal of Cell Biology 173, 937–948

Abstract: 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.  

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Shillingford JM, Murcia NS, Larson CH, Low SH, Hedgepeth R, Brown N, Flask CA, Novick AC, Goldfarb DA, Kramer-Zucker A, Walz G, Piontek KB, Germino GG, Weimbs T (2006)
The mTOR pathway is regulated by polycystin-1 and its inhibition reverses renal cystogenesis in polycystic kidney disease.
Proceedings of the National Academy of Sciences of the USA 103, 5466-5471

Abstract: Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.

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Low SH, Vasanji A, Nanduri J, He M, Sharma N, Koo M, Drazba J, Weimbs T. (2006)
Syntaxins 3 and 4 Are Concentrated in Separate Clusters on the Plasma Membrane prior to the Establishment of Cell Polarity.
Molecular Biology of the Cell 17, 977-989

Abstract: Syntaxins 3 and 4 localize to the apical and basolateral plasma membrane, respectively, of epithelial cells where they mediate vesicle fusion. Here we report that before establishment of cell polarity, syntaxins 3 and 4 are confined to mutually exclusive, submicron sized clusters. Syntaxin clusters are remarkably uniform in size, independent of expression levels, and are distinct from caveolae and clathrin coated pits. SNAP-23 partially colocalizes with both syntaxin 3 and 4 clusters. Deletion of the apical targeting signal of syntaxin 3 does not prevent sorting into clusters away from syntaxin 4. Syntaxin 3 and 4 cluster formation depends on different mechanisms because the integrity of syntaxin 3 clusters depends on intact microtubules while syntaxin 4 clusters depend on intact actin filaments. Cholesterol depletion causes dispersion of syntaxin 3 but not syntaxin 4 clusters. In migrating cells, syntaxin clusters polarize to the leading edge suggesting a role in polarized exocytosis. These results suggest that exocytosis occurs at small fusion sites exhibiting high local concentrations of SNARE proteins that may be required for efficient membrane fusion. The establishment of separate clusters for each syntaxin suggests that the plasma membrane is inherently polarized on an ultrastructural level even before the establishment of true cell polarity.

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Low SH*, Vasanth S*, Larson CH*, Mukherjee S, Sharma N, Kinter MT, Kane ME, Obara T, and Weimbs T (2006)
Polycystin-1, STAT6 and P100 function in a novel pathway that transduces ciliary mechanosensation to gene expression and is activated in polycystic kidney disease.
Developmental Cell 10, 57-69

(* equally contributing first authors)

Abstract: Primary cilia are implicated in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD), which results from defects in polycystin-1 (PC1), but the function of PC1 remains poorly understood. Here, we show that PC1 undergoes proteolytic cleavage that results in nuclear translocation of its cytoplasmic tail. The PC1 tail interacts with the transcription factor STAT6 and the coactivator P100, and it stimulates STAT6-dependent gene expression. Under normal conditions, STAT6 localizes to primary cilia of renal epithelial cells. Cessation of apical fluid flow results in nuclear translocation of STAT6. Cyst-lining cells in ADPKD exhibit elevated levels of nuclear STAT6, P100, and the PC1 tail. Exogenous expression of the human PC1 tail results in renal cyst formation in zebrafish embryos. These results identify a novel mechanism of cilia function in the transduction of a mechanical signal to changes of gene expression involving PC1 and show that this pathway is inappropriately activated in ADPKD.

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Fei, B., Flask, C., Wang, H., Pi, A., Wilson, D.L., Shillingford, J., Murcia, N., Weimbs, T., Duerk, J.L.(2005).
Image Segmentation, Registration and Visualization of Serial MR Images for Therapeutic Assessment of Polycystic Kidney Disease in Transgenic Mice
Conf Proc IEEE Eng Med Biol Soc. 1(1):467-469, 467- 469

Abstract: In vivo small animal imaging provides a powerful tool for the study of a variety of diseases. Magnetic resonance imaging (MRI) has become an established technology for the assessment of therapies. In this study, we used high-resolution MRI to evaluate polycystic kidney disease (PKD) in transgenic mice. We used a customized mouse coil to acquire serial MR images from both wide-type and transgenic PKD mice immediately prior to, and 2-week and 4-week after therapy. We developed image segmentation, registration and visualization methods for this novel imaging application. We measured the kidney volumes for each mouse to assess the efficacy of the therapy. The segmentation results show that the kidney volumes are consistent, which are 348.7 ± 19.7 mm3 for wild-type mice and 756.3 ± 44.1 mm3 for transgenic mice, respectively. The image analysis methods provide a useful tool for this new application.

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Fan. S., Hurd, T.W., Liu, C.-J., Straight, S.W., Weimbs, T., Hurd, E.A., Domino, S.E. and Ben Margolis (2004).
Polarity proteins control ciliogenesis via kinesin motor interactions.
Current Biology 14, 1451-61

Abstract: BACKGROUND: Cilia 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. RESULTS: Here 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. CONCLUSIONS: Our 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.

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Tartakoff A.M., Matera A.G., Pimplikar S.W., Weimbs T. (2004).
Regulation of nuclear functions – nucleocytoplasmic transport in context.
European Journal of Cell Biology 83, 185-192

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Weimbs, T., Low, S.H. and Li, X. (2003)
SNAREs and epithelial cells
Methods 30, 191-197

Abstract: SNARE proteins control the membrane fusion events of membrane trafficking pathways. Work in epithelial cells has shown that polarized trafficking to the apical and basolateral plasma membrane domains requires different sets of SNAREs, suggesting a mechanism that contributes to the overall specificity of polarized trafficking and, perhaps, the formation and maintenance of polarity itself. This article describes methods that have been designed and adapted specifically for the investigation of SNAREs in epithelial cells. The knowledge of the subcellular localization of a SNARE of interest is essential to understand its function. Unfortunately, the endogenous expression levels of SNAREs are often low which makes detection challenging. We provide guidelines for determination of the localization of SNAREs by immunofluorescence microscopy including methods for signal amplification, antigen retrieval, and suppression of antibody cross-reactivity. To define which trafficking pathway a SNARE of interest is involved in, one needs to specifically inhibit its function. We provide guidelines for SNARE inhibition by overexpression of the SNARE of interest. An alternative is to introduce inhibitors of SNARE function, such as antibodies or clostridial toxins, into cells. Two methods are presented to make this possible. The first allows the monitoring of effects on trafficking pathways by biochemical assays, and is based on plasma membrane permeabilization using the bacterial toxin streptolysin-O. The second is suitable for single cell observations and is based on microinjection.

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Low, S.H., Li, X., Miura, M., Kudo, N., Quinones, B. and Weimbs, T. (2003)
Syntaxin 2 and Endobrevin are Required the Terminal Step of Cytokinesis.
Developmental Cell 4, 753-759

Abstract: 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 plasmamembrane.

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Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M. and Rodriguez-Boulan, E. (2003)
Three-dimensional Analysis of Post-Golgi Carrier Exocytosis in Epithelial Cells.
Nature Cell Biology 5, 126-136


Abstract: 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 (TIRFM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFPtagged 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.

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Sherief, M.H., Low, S.H., Miura, M., Kudo, N., Novick, A.C. and Weimbs, T. (2003)
Matrix Metalloproteinase Activity in Urine of Renal Cell Carcinoma Patients Leads to Degradation of Extracellular Matrix Proteins: Possible Use as a Screening Assay.
Journal of Urology 169, 1530–1534


Abstract: Purpose: Localized renal cell carcinoma is usually curable by nephrectomy. However, a large fraction of patients already present with metastatic disease, which results in a poor outcome. Currently no clinically relevant screening assay is available to detect early stage renal cell carcinoma. We investigated whether urinary extracellular matrix (ECM) proteins and/or matrix metalloproteinase (MMP) activity may be valuable as a noninvasive indicator of early stage renal cell carcinoma.
Materials and Methods: Urine specimens from preoperative patients with renal cell carcinoma and healthy controls were collected. The urinary excretion of the ECM proteins collagen IV, laminin and fibronectin was investigated by immunoblotting. MMP activity was assessed by gelatin zymography and by a fluorescence based microtiter plate activity assay.
Results: The full-length forms of all 3 ECM proteins investigated were significantly decreased or absent in renal cell carcinoma urine. Based on criteria established in this study this finding would lead to the correct detection of 95% of patients with renal cell carcinoma (21 of 22) with a false-positive rate of 4.5% (1 of 22 controls). All 11 nonmetastatic cases of the lowest clinical stage (T1N0M0) were correctly identified. The absence of urinary ECM proteins was due to significantly increased urinary MMP activity.
Conclusions: Analysis of decreased urinary ECM proteins and analysis of increased MMP activity may have value for the development of a sensitive, high throughput molecular screening assay to detect early stage renal cell carcinoma.

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Low, S.H., Marmorstein, L.Y, Miura, M., Li, X., Kudo, N., Marmorstein, A.D. and Weimbs, T. (2002)
Retinal pigment epithelial cells exhibit unique expression and localization of plasma membrane syntaxins which may contribute to their trafficking phenotype.
Journal of Cell Science 115, 4545-4553


Abstract: The SNARE membrane fusion machinery controls the fusion of transport vesicles with the apical and basolateral plasma-membrane domains of epithelial cells and is implicated in the specificity of polarized trafficking. To test the hypothesis that differential expression and localization of SNAREs may be a mechanism that contributes to cell type-specific polarity of different proteins, we studied the expression and distribution of plasma-membrane SNAREs in the retinal pigment epithelium (RPE), an epithelium in which the targeting and steady-state polarity of several plasma membrane proteins differs from most other epithelia. We show here that retinal pigment epithelial cells both in vitro and in vivo differ significantly from MDCK cells and other epithelial cells in their complement of expressed t-SNAREs that are known – or suggested – to be involved in plasma membrane trafficking. Retinal pigment epithelial cells lack expression of the normally apical-specific syntaxin 3. Instead, they express syntaxins 1A and 1B, which are normally restricted to neurons and neuroendocrine cells, on their apical plasma membrane. The polarity of syntaxin 2 is reversed in retinal pigment epithelial cells, and it localizes to a narrow band on the lateral plasma membrane adjacent to the tight junctions. In addition, syntaxin 4 and the v-SNARE endobrevin/ VAMP-8 localize to this sub-tight junctional domain, which suggests that this is a region of preferred vesicle exocytosis. Altogether, these data suggest that the unique polarity of many retinal pigment epithelial proteins results from differential expression and distribution of SNAREs at the plasma membrane. We propose that regulation of the expression and subcellular localization of plasma membrane SNAREs may be a general mechanism that contributes to the establishment of distinct sorting phenotypes among epithelial cell types.

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Li, X., Low, S.H., Miura, M. and Weimbs, T. (2002)
Differential SNARE Expression and Localization in Renal Epithelial Cells suggests mechanism for establishment of distinct trafficking phenotypes.
American Journal of Physiology 283: F1111–F1122


Abstract: The apical- and basolateral- specific distribution of target soluble N-ethylmaleimide- sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in alpha- but not beta-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

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van IJzendoorn SC, Tuvim MJ, Weimbs T, Dickey BF, Mostov KE. (2002).
Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells.
Developmental Cell 2, 219-28

Abstract: 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.

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Lim, S. N., Bonzelius, F., Low, S. H., Wille, H., Weimbs, T., and Herman, G. A. (2001).
Identification of discrete classes of endosome-derived small vesicles as a major cellular pool for recycling membrane proteins.
Molecular Biology of the Cell 12, 981-95

Abstract: Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5'-[gamma-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

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Low, S. H., Miura, M., Roche, P. A., Valdez, A. C., Mostov, K. E., and Weimbs, T. (2000)
Intracellular re-direction of plasma membrane trafficking after loss of epithelial cell polarity.
Molecular Biology of the Cell 11, 3045-3060

Abstract: In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin- Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.

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S. H. Low, S. J. Chapin, C. Wimmer, S. W. Whiteheart, L. K. Kömüves, K. E. Mostov, and T. Weimbs (1998).
The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells.
Journal of Cell Biology 141, 1503-1513

Abstract: We have investigated the controversial involvement of components of the SNARE machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 over-expression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against a-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of NSF in TGN to apical transport while basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23 and a-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism which is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery which suggests that they play a role in providing specificity to polarized membrane traffic.

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T. Weimbs, K. E. Mostov, S. H. Low and K. Hofmann (1998)
A model for structural similarity between different SNARE complexes based on sequence relationships
Trends in Cell Biology 8, 260-262

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S. H. Low, P. A. Roche, H. A. Anderson, S. C. D. van IJzendoorn, M. Zhang, K. E. Mostov, and T. Weimbs, (1998).
Targeting of SNAP-23 and SNAP-25 in Polarized Epithelial Cells
Journal of Biological Chemistry 273, 3422-3430

ABSTRACT: SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.

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T. Weimbs, S. H. Low, S. J. Chapin, and K. E. Mostov (1997)
Apical targeting in polarized epithelial cells: there's more afloat than rafts
Trends in Cell Biology 7, 393-399

ABSTRACT: Most metazoan cells are 'polarized'. One crucial aspect of this polarization is that the plasma membrane is divided into two or more domains with different protein and lipid compositions - for 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, which together promise at least a partial understanding of the underlying general mechanisms that ensure the necessary specificity of these pathways.

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T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher and K. Hofmann (1997)
A conserved domain is present in different families of vesicular fusion proteins: A new superfamily
Proc. Natl. Acad. Sci. USA 94, 3046-3051

ABSTRACT: We have analyzed conserved domains in t-SNAREs, proteins which are believed to be involved in the fusion of transport vesicles with their target membrane. Using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain which is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 families, which therefore form a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain was also identified in several proteins which have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode and mammalian proteins were identified which belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins.

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S. H. Low*, S. J. Chapin*, T. Weimbs*, L. G. Kömüves, M. K. Bennett and K. E. Mostov (1996)
Differential Localization of Syntaxin Isoforms in Polarized MDCK Cells
Molecular Biology of the Cell 7, 2007-2018
(*equal first authorship)

ABSTRACT: Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3 and 4 were found to be endogenously present in Madin-Darby canine kidney (MDCK) cells. The localization of syntaxins 1A, 1B, 2, 3 and 4 in stably transfected MDCK cell lines was studied by confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, while the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.

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K. E. Mostov, Y. Altschuler, S. J. Chapin, C. Enrich, S. H. Low, F. Luton, J. Richman- Eisenstat, K. L. Singer, K. Tang and T. Weimbs (1995)
Regulation of Protein Traffic in Polarized Epithelial Cells: The Polymeric Immunoglobulin Receptor Model
Cold Spring Harb. Symp. Quant. Biol. 60, 775-781

T. Weimbs and W. Stoffel (1994)
Topology of CNS myelin proteolipid protein: evidence for the nonenzymatic glycosylation of extracytoplasmic domains in normal and diabetic animals
Biochemistry 33, 10408-15

ABSTRACT: Myelin proteolipid protein (PLP), the main integral membrane protein in the central nervous system myelin, was labeled at the extracytoplasmic domains with the membrane impermeant reagents pyridoxal 5'-phosphate and tritiated borohydride. Lysine- 217, located in the fourth hydrophilic domain of PLP, was found to be the major labeled residue, which defined this domain to be extracytoplasmic in agreement with our previously proposed topological model. The remarkably high reactivity in vitro of this residue as compared to all other lysines in PLP led us to investigate the possible modification of PLP in vivo by other carbonyl compounds. We demonstrate that PLP is the most highly nonenzymatically glycosylated membrane protein in murine and bovine brain. The degree of modification increases significantly under hyperglycemic conditions, as studied in diabetic mice. The majority of the glycosylation sites are also located at extracytoplasmic domains. The degree of nonenzymatic glycosylation of PLP may be related to late diabetic complications affecting the central nervous system.

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T. Weimbs and W. Stoffel (1992)
Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP
Biochemistry 31, 12289-96

ABSTRACT: Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I- AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.

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T. Weimbs, T. Dick, W. Stoffel and E. Boltshauser (1990)
A point mutation at the X-chromosomal proteolipid protein locus in Pelizaeus-Merzbacher disease leads to disruption of myelinogenesis
Biological Chemistry Hoppe Seyler 371, 1175-83

ABSTRACT: A group of inherited neurological disorders are the X-chromosome linked dysmyelinoses, in which myelin membranes of the CNS are missing or perturbed due to a strongly reduced number of differentiated oligodendrocytes. In animal dysmyelinoses (jimpy mouse, msd-mouse, md rat, shaking pup) mutations of the main integral myelin membrane protein, proteolipid protein, have been identified. Pelizaeus-Merzbacher disease (PMD) or sudanophilic leucodystrophy is an X-linked dysmyelinosis in humans. We report here on the molecular basis of the defect of affected males of a PMD kindred. Rearrangements of the PLP gene were excluded by Southern blot hybridisation analysis and PCR amplification of overlapping domains of the PLP gene. Sequence analysis revealed one single C----T transition in exon IV, which leads to a threonine----isoleucine substitution within a hydrophobic intramembrane domain. The impact of this amino-acid exchange on the structure of PLP in the affected cis membrane domain is discussed. A space filling model of this domain suggests a tight packing of the alpha-helices of the loop which is perturbed by the amino-acid substitution in this PMD exon IV mutant. The C----T transition in exon IV abolishes a Hph I restriction site. This mutation at the recognition site for Hph I (RFLP) and allele-specific primers have been used for mutation screening the PMD kindred.

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