Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement

摘要

class="head no_bottom_margin" id="sec1title">Results and DiscussionTo 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 (phaeochromocytoma cells, PC12s) using soluble cargo Neuropeptide Y (NPY) fused to EGFP [, ]. We stimulated cells to secrete and then followed the recruitment of new LDCVs using total internal reflection fluorescence microscopy (TIRFM). We quantified trajectories taken by all vesicles before, during, and after exocytosis. 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. B displays frames from , showing a vesicle arriving on top of a site occupied by an earlier LDCV, scanning the same region before moving off to visit at least two other previously occupied regions. The area under scrutiny is ∼4 μm². We found LDCVs follow similar trajectories to similar (but not identical) fusion sites on the cell surface (A–S2D), even after treatment with Methyl-β-cyclodextrin to quantifiably disrupt plasma membrane lipid order [] (E–S2G). These data appear to support current models, suggesting that LDCVs visit preferred sites on the membrane, using defined, re-usable routes that intuitively appear like physical tracks.Current Model for Vesicle Dynamics at the Membrane(A) Stylized cartoon showing a model for vesicle dynamics at the plasma membrane, where LDCVs are docked on molecular machinery depots. This is illustrated as a “well,” attracting LDCVs.(B) Images from a TIRFM recording of a PC12 cell expressing lumenal NPY-EGFP. A single vesicle (gray sphere) is shown (track in color) scanning the membrane, visiting areas preferred by other vesicles (gray tracks). Grid scale, 500 nm grid edge. Color bar shows time.See also and .