分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-11
摘要: Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (similar to 10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of preformed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5 beta/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: Background: The recent developments of far-field optical microscopy (single molecule imaging techniques) have overcome the diffraction barrier of light and improve image resolution by a factor of ten compared with conventional light microscopy. These techniques utilize the stochastic switching of probe molecules to overcome the diffraction limit and determine the precise localizations of molecules, which often requires a long image acquisition time. However, long acquisition times increase the risk of sample drift. In the case of high resolution microscopy, sample drift would decrease the image resolution. Results: In this paper, we propose a novel metric based on the distance between molecules to solve the drift correction. The proposed metric directly uses the position information of molecules to estimate the frame drift. We also designed an algorithm to implement the metric for the general application of drift correction. There are two advantages of our method: First, because our method does not require space binning of positions of molecules but directly operates on the positions, it is more natural for single molecule imaging techniques. Second, our method can estimate drift with a small number of positions in each temporal bin, which may extend its potential application. Conclusions: The effectiveness of our method has been demonstrated by both simulated data and experiments on single molecular images.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Background: Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Results: Superresolution imaging reveals homogeneous distribution of PI(4,5)P-2, PI4P, and PI(3,4,5)P-3 in the major area of the PM. Conclusion: Phosphatidylinositides detected by PH domains are uniformly distributed in the major regions of the PM, with limited concentration gradients. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale. Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P-2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P-2-enriched membrane patches/domains of diverse sizes (383 +/- 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P-2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P-3) showed similar spatial distributions as PI(4,5)P-2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.