Abstract
The role of cytoskeletal elements in regulating transport and docking steps that precede exocytosis of secretory organelles is not well understood. We have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the three-dimensional motions of secretory organelles near the plasma membrane in living endothelial cells. Weibel-Palade bodies (WPb), the large tubular storage organelles for von Willebrand factor, were labelled with Rab27a-GFP. By contrast, green fluorescent protein (GFP)-tagged tissue-type plasminogen activator (tPA-GFP) labelled submicron vesicular organelles. Both populations of GFP-labelled organelles underwent stimulated exocytosis. The movement of these morphologically distinct organelles was measured within the evanescent field that penetrated the first 200 nm above the plasma membrane. WPb and tPA-GFP vesicles displayed long-range bidirectional motions and short-range diffusive-like motions. Rotating and oscillating WPb were also observed. TIRF microscopy enabled us to quantify the contribution of actin and microtubules and their associated motors to the organelle motions close to the plasma membrane. Long-range motions, as well as WPb rotations and oscillations, were microtubule-and kinesin-dependent. Disruption of the actin cytoskeleton and inhibition of myosin motors increased the number of long-range motions and, in the case of WPb, their velocity. The actin and microtubules had opposite effects on the mobility of organelles undergoing short-range motions. Actin reduced the mobility and range of motion of both WPb and tPA vesicles, whereas microtubules and kinesin motors increased the mobility of WPb. The results show that the dynamics of endothelial secretory organelles close to the plasma membrane are controlled by the opposing roles of the microtubule and actin cytoskeletal transport systems.
Highlights
Microtubules, actin filaments and their associated molecular motors drive the movement of intracellular organelles
The cytoskeleton is involved in late transport steps and subsequent docking to and fusion with the plasma membrane, most studies have focused on the role of the actin cortex, which is thought to act as a barrier preventing organelle motion and docking to the plasma membrane (Valentijn et al, 1999)
TPA-green fluorescent protein (GFP) localized to small-diameter vesicles
Summary
Microtubules, actin filaments and their associated molecular motors drive the movement of intracellular organelles. In ‘sequential’ models, fast long-range transport is mediated by microtubules and actin provides short-range local transport or restricts the organelle movement at its final destination (Goode et al, 2000; Rogers and Gelfand, 2000; Wu et al, 2000). By contrast, ‘tug-of-war’ models assume that different types of motors act simultaneously and that the motion of an organelle results from a balance of the forces exerted on the organelle (Gross et al, 2002a; Gross et al, 2002b). In the case of secretory organelles, the final destination is the plasma membrane, where the organelles undergo exocytosis. Less is understood about the role of microtubules and their interaction with the actin cytoskeleton to regulate the transport and motion of secretory organelles close to the plasma membrane
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
