The function of the nervous system relies on synaptic transmission. Synaptic transmission is mediated by calcium-triggered vesicle fusion, followed by vesicle endocytosis that recycles vesicles. Although significant progress has been made in understanding these processes, much remains unknown. My goal is to advance our understanding of these synaptic signaling processes. The progress of the last year is described below. 1. For decades, two fusion modes were thought to control hormone and transmitter release essential to life: one facilitates release via fusion pore dilation and flattening (full-collapse), the other limits release by closing a narrow fusion pore (kiss-and-run). Using super-resolution STED microscopy to visualize fusion modes in neuroendocrine cells, we found that facilitation of release was not mediated by full-collapse, but a fusion mode in which fused vesicles shrank. Equally surprising, inhibition of release was not mediated by kiss-and-run, but a fusion mode in which fused vesicles grew. These findings challenge the traditional view that transmitter release is controlled by full-collapse and kiss-and-run fusion mode. A manuscript about this story has been submitted to a journal for peer review. 2. Calcium influx facilitates vesicle mobilization to the readily releasable pool, which provides vesicles for exocytosis and thus maintains synaptic transmission during repetitive firing. How calcium influx facilitates vesicle mobilization is poorly understood. Here we found that protein kinase C and calmodulin may serve as calcium sensors to mediate this process, which shed light on how synaptic transmission and neuronal network activity are maintained during repetitive firing. 3. Atomic force microscopy (AFM) has been used to measure cellular stiffness at different osmolarities to investigate the effect of osmotic pressure on cells. However, substantial direct evidence is essential to clarify the phenomena derived from the experimental results. This study used both the single-point and force mapping methods to measure the effective Young's modulus of the cell by using temporal and spatial information. The single-point force measurements confirmed the positive correlation between cellular stiffness and osmolarity. The force mapping measurements provided local stiffness on the cellular surface and identified the cytoskeleton distribution underneath the plasma membrane. At hyper-osmolarity, the cytoskeleton was observed to cover most of the area underneath the plasma membrane, and the effective Young's modulus on the area with cytoskeleton support was determined to be higher than that at iso-osmolarity. The overall increase in cellular Young's modulus confirmed the occurrence of cytoskeleton compression at hyper-osmolarity. On the other hand, although the average Young's modulus at hypo-osmolarity was lower than that at iso-osmolarity, we observed that the local Young's modulus measured on the areas with cytoskeleton support remained similar from iso-osmolarity to hypo-osmolarity. The reduction of the average Young's modulus at hypo-osmolarity was attributed to reduced cytoskeleton coverage underneath the plasma membrane. 4. Reprogramming of cancers into normal-like tissues is an innovative strategy for cancer treatment. Recent reports demonstrate that defined factors can reprogram cancer cells into pluripotent stem cells. Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor in humans. Despite multimodal therapy, the outcome for patients with GBM is still poor. Therefore, developing novel therapeutic strategy is a critical requirement. We have developed a novel reprogramming method that uses a conceptually unique strategy for GBM treatment. We screened a kinase inhibitor library to find which candidate inhibitors under reprogramming condition can reprogram GBM cells into neurons. The induced neurons are identified whether functional and loss of tumorigenicity. We have found that mTOR and ROCK kinase inhibitors are sufficient to reprogram GBM cells into neural-like cells and normal neurons. The induced neurons expressed neuron-specific proteins, generated action potentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis showed that the induced neurons had a profile different from GBM cells and were similar to that of control neurons induced by established methods. In vitro and in vivo tumorigenesis assays showed that induced neurons lost their proliferation ability and tumorigenicity. Moreover, reprogramming treatment with ROCK-mTOR inhibitors prevented GBM local recurrence in mice. This study indicates that ROCK and mTOR inhibitors-based reprogramming treatment prevents GBM local recurrence. Currently ROCK-mTOR inhibitors are used as anti-tumor drugs in patients, so this reprogramming strategy has significant potential to move rapidly toward clinical trials.
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