Garry P. Morgan

High-Resolution 3D Quantitative Analysis of Caveolar Ultrastructure and Caveola–Cytoskeleton Interactions

Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure–function relationships in three‐dimension (3D) at high resolution both in conventionally fixed and in fast‐frozen/freeze‐substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3‐L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.

Islet Cholesterol Accumulation Due to Loss of ABCA1 Leads to Impaired Exocytosis of Insulin Granules

OBJECTIVE The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from β-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking β-cell ABCA1. RESEARCH DESIGN AND METHODS Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze β-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated. RESULTS We show that a lack of β-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in β-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of β-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in β-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion. CONCLUSIONS Our data highlight a crucial role of ABCA1 and cellular cholesterol in β-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired β-cell function in diabetes.

The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1–8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.

Electron Tomography Reveals Rab6 Is Essential to the Trafficking of trans-Golgi Clathrin and COPI-Coated Vesicles and the Maintenance of Golgi Cisternal Number

We have shown previously that Rab6, a small, trans‐Golgi‐localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high‐resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans‐Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin‐coated vesicles and the smaller to coat protein I (COPI)‐coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans‐Golgi/trans‐Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6‐depleted HeLa cells that we observed may well be coupled to the trans‐Golgi accumulation of COPI‐coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6‐dependent protein machine affecting Golgi‐proximal, coated vesicle accumulation and probably transport at the trans‐Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

The caveolin–cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle

The caveolar membrane microdomain plays an integral role in stabilizing the muscle fiber surface in mice and zebrafish.

Structural Insights into the Organization of the Cavin Membrane Coat Complex

Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.

Structural studies of Planctomycete Gemmata obscuriglobus support cell compartmentalisation in a bacterium

Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.