Abstract
Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis.
Highlights
The directional transfer of membrane and soluble proteins from one cellular compartment to another is essential for cell survival
All of these were readily detected by reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from polarized MDCK cells (Figure 1)
Individual SNARE proteins localize to distinct plasma membrane subdomains of polarized epithelial cells to provide specificity in vesicle fusion with these domains
Summary
The directional transfer of membrane and soluble proteins from one cellular compartment to another is essential for cell survival. A critical step in these membrane trafficking events is the selective fusion of vesicles with target organelles. SNAREs (Soluble Nethylmaleimide-sensitive factor Attachment protein REceptors) are key components of the machinery required to maintain selectivity, and are directly responsible for fusion. These small proteins localize to organelle and vesicle membranes and interact when the two membranes are in close proximity. One R-SNARE on the vesicle membrane and three Q-SNAREs on the target membrane interact to form a helical bundle. A subset of R-SNAREs fall into a family known as VAMPs (Vesicle Associated Membrane Proteins), based on their initial discovery in synaptic vesicle membranes
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