An imaging toolbox for understanding the molecular biology of calcium triggered vesicle fusion in situ

Calcium ions in the human body are responsible for cell homeostasis. In addition, many cell functions, such as synaptic transmission, hormone and neuropeptide release are triggered by calcium. Ca2+ concentration must be therefore carefully regulated by voltagegated calcium channels. Exocytosis is the process of the fusion of the secretory vesicle with the plasma membrane. For effective and fast exocytosis, vesicles must be docked near calcium channels. The probability of the release of synaptic vesicles is hypothesized to increase with the number of proximal calcium channels. N-type (Cav2.2) calcium channels cooperate directly with SNARE proteins and synaptotagmin through the specific synprint site, which is located on the main pore forming subunit. In recent years the amount of research on calcium channels has markedly increased, but there are still limitations in the methods used to study Ca2+. This results in a loss of information with regard to the true location of a point source that is emitting light and therefore the proteins we want to localize. The development of super-resolution techniques allows imaging of these proteins closer to the molecular scale, enabling us to better understand these cellular processes. The results presented in this thesis aim to understand the distribution and behaviour of Ntype calcium channels across the cell membrane, using advanced microscopy techniques to image below the diffraction limit. Our findings revealed that the synprint site has an influence on Cav2.2 calcium channel cluster patterning and behaviour. The results demonstrate no direct interaction between Cav2.2 calcium channels and syntaxin-1A. The experiments with a genetically encoded calcium indicator fused to a SNARE protein together with TIRF microscopy present a promising method to examine the calcium “activity” across the plasma membrane. Findings presented in this thesis introduce a new angle of looking at the interaction of syntaxin-1A with the synprint motif of Cav2.2 calcium channels. Taken together the results can create a novel model of the distribution and behaviour of Cav2.2 calcium channels in the secretory cells and it is recommended that these methods are employed more widely in the future to investigate ion channel distribution and function in cells

Postmortem changes in brain cell structure: a review

Brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. Following the global loss of blood flow to the brain that initiates the postmortem interval (PMI), cells rapidly become depleted of energy and begin to decompose. To ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the PMI. We searched multiple databases to identify studies measuring the effects of PMI on the morphometry (i.e. external dimensions) of brain cells. We screened 2119 abstracts, 361 full texts, and included 172 studies. Mechanistically, fluid shifts causing cell volume alterations and vacuolization are an early event in the PMI, while the loss of the ability to visualize cell membranes altogether is a later event. Decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. Geometrically, deformations of cell membranes are common early events that initiate within minutes. On the other hand, topological relationships between cellular features appear to remain intact for more extended periods. Taken together, there is an uncertain period of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. This review may be helpful for investigators studying human postmortem brain tissue, wherein the PMI is an unavoidable aspect of the research

Postmortem changes in brain cell structure: a review

Brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. Following the global loss of blood flow to the brain that initiates the postmortem interval (PMI), cells rapidly become depleted of energy and begin to decompose. To ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the PMI. We searched multiple databases to identify studies measuring the effects of PMI on the morphometry (i.e. external dimensions) of brain cells. We screened 2119 abstracts, 361 full texts, and included 172 studies. Mechanistically, fluid shifts causing cell volume alterations and vacuolization is an early event in the PMI, while loss of cell membrane visualization altogether is a later event. Decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. Geometrically, deformations of cell membranes are common early events that initiate within minutes. On the other hand, topological relationships between cellular features appear to be intact for more extended periods. Taken together, there is an uncertain length of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. This review may be helpful for investigators studying human postmortem brain tissue, wherein the PMI is an unavoidable aspect of the research

Navigation through the plasma membrane molecular landscape shapes random organelle movement

Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9–13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle “pools” are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane

A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-β2 signaling

Many breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGAP1low cells display a motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells in the lungs of mice bearing BC tumors. Overall, srGAP1low cells have increased Smad2 activation and TGF-β2 secretion, resulting in increased invasion and p27 levels to sustain quiescence. These findings identify srGAP1 as a mediator of a proliferative to invasive phenotypic switch in BC cells in vivo through a TGF-β2-mediated signaling axis