Mutations in the channel protein PKD2 cause autosomal dominant polycystic kidney disease, but the function of PKD2 in cilia remains unclear. Here, we show that PKD2 targets and anchors mastigonemes, filamentous polymers of the glycoprotein MST1, to the extracellular surface of Chlamydomonas cilia. PKD2-mastigoneme complexes physically connect to the axonemal doublets 4 and 8, positioning them perpendicular to the plane of ciliary beating.
View Article and Find Full Text PDFThe resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments.
View Article and Find Full Text PDFExocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca sensor that penetrates membranes upon binding Ca to trigger synchronous vesicle fusion.
View Article and Find Full Text PDFThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state.
View Article and Find Full Text PDFMisfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson's disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear.
View Article and Find Full Text PDFSNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly conserved set of membrane-associated proteins that mediate intracellular membrane fusion. Cognate SNAREs from two separate membranes zipper to facilitate membrane apposition and fusion. Though the stable post-fusion conformation of SNARE complex has been extensively studied with biochemical and biophysical means, the pathway of SNARE zippering has been elusive.
View Article and Find Full Text PDFC2 domains are widespread motifs that often serve as Ca(2+)-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca(2+) sensor for neuronal exocytosis.
View Article and Find Full Text PDFLipid molecules, structural components of biomembranes, have been proposed for an important role in membrane fusion. Through various techniques based on a protein-reconstituted vesicle-vesicle fusion system, we investigated the influence of several lipid molecules on different stages of a yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion process. Lipid compositions played a significant role in the early stages, docking and lipid mixing, while only exhibiting a minor effect on fusion pore formation and dilation phases, indicated by both small and large content mixing.
View Article and Find Full Text PDFSynaptotagmin 1 (Syt1), a major Ca(2+) sensor in neuroexocytosis, utilizes SNARE- and membrane-binding to regulate vesicle fusion, a required process for neurotransmitter release at the synapse. However, the mechanism by which Syt1 orchestrates SNARE- and membrane- binding to control individual vesicle fusion steps is still unclear. In this study, we used a number of single vesicle assays that can differentiate intermediates of neuroexocytosis, to focus on Syt1 mutants that might impair Syt1-SNARE/PIP2 interaction, Ca(2+)-binding, or membrane penetration.
View Article and Find Full Text PDFIn neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca(2+) sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca(2+)-triggered content mixing by Munc18-1 are observed.
View Article and Find Full Text PDFα-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear.
View Article and Find Full Text PDFSynaptotagmin 1 (Syt1) is a major Ca(2+)-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca(2+), indicating C2AB does not interfere with the initial nucleation of SNARE assembly.
View Article and Find Full Text PDFSNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized, pre-fusion conformations have been elusive. We used single-molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes.
View Article and Find Full Text PDFSyt1 (synaptotagmin 1), a major Ca2+ sensor for fast neurotransmitter release, contains tandem Ca2+-binding C2 domains (C2AB), a single transmembrane α-helix and a highly charged 60-residue-long linker in between. Using single-vesicle-docking and content-mixing assays we found that the linker region of Syt1 is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker contains the basic-amino-acid-rich N-terminal region and the acidic-amino-acid-rich C-terminal region.
View Article and Find Full Text PDFThioredoxin (Trx) and glutathione/glutaredoxin (GSH/Grx) systems play the dominant role in cellular redox homeostasis. Recently the Trx system has been shown to be responsible to control the balance of GSH/GSSG once the glutathione reductase system is not available. To decipher the structural basis of electron transfer from the Trx system to GSSG, we solved the crystal structures of oxidized Trx1 and glutathionylated Trx1Cys33Ser mutant at 1.
View Article and Find Full Text PDFActa Crystallogr Sect F Struct Biol Cryst Commun
February 2009
Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.
View Article and Find Full Text PDFActa Crystallogr Sect F Struct Biol Cryst Commun
January 2009
Thioredoxins (Trxs) are a family of small redox-active proteins that are found in all living organisms. In Saccharomyces cerevisiae, two cytosolic Trxs (Trx1 and Trx2) and one mitochondrial Trx (Trx3) have previously been identified. In this work, cytosolic Trx1 containing a C33S mutant was overexpressed, purified, glutathionylated and crystallized using the hanging-drop vapour-diffusion method.
View Article and Find Full Text PDFBiomed Environ Sci
June 2008
Objective: To examine the neuroprotective effects of a novel manganese porphyrin, manganese (III) meso-tetrakis (N,N'-diethylimidazolium-2-yl) porphyrin (MnTDM), in the mouse model of Parkinson's disease (PD) induced by paraquat (PQ).
Methods: Male C57BL/6 mice were subcutaneously injected with either saline or PQ at 2-day intervals for a total of 10 doses, MnTDM was subcutaneously injected with the PQ 2 h before treatment. Performance on the pole and swim test were measured 7 days after the last injection and animals were sacrificed one day later.