Abstract
SummaryEukaryotic 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, 10, 11, 12, 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.
Highlights
We previously used photoactivated localization microscopy (PALM) [20, 21] and direct stochastic optical reconstruction microscopy [22] to relate syntaxin1a and SNAP-25 molecular positions with those of single large dense-core vesicles (LDCVs) [4, 5, 18]
To address the question of whether membrane-proximal vesicles behave in a controlled manner, we first posed a simple question: when new vesicles are recruited to the plasma membrane, is this spatially random, or is there some order to this process? We labeled large dense-core vesicles (LDCVs) in secretory cells using soluble cargo Neuropeptide Y (NPY) fused to EGFP [17, 18]
This marked the image plane of the plasma membrane with areas visited by LDCVs during the recording period and allowed us to determine the arrival sites of any new recruits
Summary
We previously used photoactivated localization microscopy (PALM) [20, 21] and direct stochastic optical reconstruction microscopy (dSTORM) [22] to relate syntaxin1a and SNAP-25 molecular positions with those of single LDCVs [4, 5, 18]. For this we used fluorescence correlation spectroscopy, as we have before [4], in primary cells and cell lines, finding no evidence of aggregation of fusions or unfused mCherr
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