Project description:The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.
Project description:Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.
Project description:The interior of eukaryotic cells is mysterious. How do the large communities of macromolecular machines interact with each other? How do the structures and positions of these nanoscopic entities respond to new stimuli? Questions like these can now be answered with the help of a method called electron cryotomography (cryo-ET). Cryo-ET will ultimately reveal the inner workings of a cell at the protein, secondary structure, and perhaps even side-chain levels. Combined with genetic or pharmacological perturbation, cryo-ET will allow us to answer previously unimaginable questions, such as how structure, biochemistry, and forces are related in situ. Because it bridges structural biology and cell biology, cryo-ET is indispensable for structural cell biology-the study of the 3-D macromolecular structure of cells. Here we discuss some of the key ideas, strategies, auxiliary techniques, and innovations that an aspiring structural cell biologist will consider when planning to ask bold questions.
Project description:Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans-Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3P)-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.
Project description:During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a highly ordered sequence of events starting with the detachment of tumor cells from the extracellular matrix (E.C.M.). In normal cells, detachment from E.C.M. triggers programmed cell death, termed anoikis. However, tumor cells dodge their way to anoikis and spread to distant sites for initiating the metastatic program. In this work, we explored the impact of E.C.M. detachment on the expression of some major oncogenic histone methyltransferases. Results showed both EZH2 expression and its enzymatic activity were significantly increased in E.C.M. detached cancer cells when compared to the attached cells. Inhibition of EZH2 results in a significant reduction in cell proliferation, spheroids size, and induction in apoptosis in E.C.M. detached cells. Furthermore, we observed a reduction in EZH2 expression levels in single cells when compared to clusters of E.C.M. detached cells. Finally, we combined the EZH2 inhibition with AMPK, known to be highly expressed in E.C.M. detached cancer cells and observed antagonistic effects between the two pathways. The observed results clearly showed that E.C.M. detached cancer cells require oncogenic EZH2 and can be targeted by EZH2 inhibitors.
Project description:Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.
Project description:The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.
Project description:Step-growth, radically mediated thiol-norbornene photopolymerization is used to create versatile, stimuli-responsive poly(ethylene glycol)-co-peptide hydrogels The reaction is cytocompatible and allows for the encapsulation of human mesenchymal stem cells with a viability greater than 95%. Cellular spreading is dictated via three-dimensional biochemical photopatterning.
Project description:Cryo-electron tomography (cryo-ET) is one of the most advanced technologies for the in situ visualization of molecular machines by producing three-dimensional (3D) biological structures. However, cryo-ET imaging has two serious disadvantages-low dose and low image contrast-which result in high-resolution information being obscured by noise and image quality being degraded, and this causes errors in biological interpretation. The purpose of this research is to explore an optimal wavelet denoising technique to reduce noise in cryo-ET images. We perform tests using simulation data and design a filter using the optimum selected wavelet parameters (three-level decomposition, level-1 zeroed out, subband-dependent threshold, a soft-thresholding and spline-based discrete dyadic wavelet transform (DDWT)), which we call a modified wavelet shrinkage filter; this filter is suitable for noisy cryo-ET data. When testing using real cryo-ET experiment data, higher quality images and more accurate measures of a biological structure can be obtained with the modified wavelet shrinkage filter processing compared with conventional processing. Because the proposed method provides an inherent advantage when dealing with cryo-ET images, it can therefore extend the current state-of-the-art technology in assisting all aspects of cryo-ET studies: visualization, reconstruction, structural analysis, and interpretation.