Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

Trexler, Adam J.; Sochacki, Kem A.; Taraska, Justin W.

doi:10.1091/mbc.e16-01-0057

Cited by 44 publications

(92 citation statements)

References 85 publications

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“…This supports the existing idea that vesicles can be co-transported in clusters [7]. In a recent study in insulin-secreting INS-1 cells, tomosyn tightly associated with NPY-GFP labeled DCVs where it remained associated until near the time of vesicle fusion and then diffused away [62]. Taken together, these observations support the hypothesis that tomosyn contributes to the trafficking and delivery of secretory vesicles.…”

Section: Discussionsupporting

confidence: 85%

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

et al. 2017

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The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.

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Section: Discussionsupporting

confidence: 85%

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

et al. 2017

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“…pHluorin fluorescence is quenched in the acidic lumen of the vesicle, but upon fusion the lumenal pH is neutralized by the extracellular buffer causing pHluorin signal to dramatically increase, enabling the detection of exocytic events. To stimulate exocytosis, we depolarized cells by applying buffer containing high extracellular KCl using a superfusion pipette positioned close to the cell (Trexler et al, 2016;Trexler and Taraska, 2017). Membrane depolarization caused a substantial increase in the frequency of fusion events as shown in Supplemental Figure 1A.…”

Section: Resultsmentioning

confidence: 99%

“…Aside from the interest in their signaling functions in endocrine cells, these vesicles have been used as experimentally tractable surrogates for the study of SV behavior (de Wit et al, 2001;Brauchi et al, 2008;Sochacki et al, 2012). Here, we used total internal reflection fluorescence (TIRF) microscopy to monitor the dynamics of over two dozen proteins during calcium-triggered exocytosis of single SLMVs in PC12 cells (Sochacki et al, 2012;Trexler et al, 2016). In an imaging-based screen examining key exocytic and endocytic proteins, we show that many proteins, including syntaxin, VAMP, tomosyn, and Rab GTPases are pre-clustered at fusion sites and rapidly diffuse away following exocytosis.…”

Section: Introductionmentioning

confidence: 99%

Local Protein Dynamics during Microvesicle Exocytosis in Neuroendocrine Cells

Somasundaram

Taraska

2018

Preprint

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Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of accessory proteins at the moments surrounding exocytosis. Our experiments reveal that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are preclustered at fusion sites and rapidly lost at fusion. The ATPase NSF is recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow down the release of vesicle membrane-bound cargo. A similar effect on cargo release was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis regulate exocytosis of synaptic-like microvesicles, possibly by modulating the highly curved neck of the vesicle fusion pore. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key modulators of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

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“…In addition, observed covariates, here whether a cell came from a healthy or diabetic donor, can be included for example in a proportional hazards formulation. We envisage that our approach to the study of exocytosis with the use of flexible survival modeling [12] can be extended to include more complicated, time-dependent covariates [45], such as for example Ca 2+ concentrations [46, 47] or protein levels [5, 7, 48] at the granules. Further extensions could take into consideration spatial information in addition to the temporal data [49].…”

Section: Discussionmentioning

confidence: 99%

Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic β-cells

et al. 2016

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Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic β-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.

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Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

Cited by 44 publications

References 85 publications

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains

Local Protein Dynamics during Microvesicle Exocytosis in Neuroendocrine Cells

Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic β-cells

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