Actin-based vesicle dynamics and exocytosis during wound wall formation in characean internodal cells

Characean internodal cells readily form wound walls upon local membrane damage. In the present study we documented the dynamics of vesicles involved in wound wall secretion and compared them with actin organization in equivalent cells using immunofluorescence. Single exocytotic events (spreading of vesicle contents) could be visualized using image enhancement by video microscopy. In control unwounded cells vesicles moved unidirectionally along parallel actin bundles and rarely contacted the plasma membrane. The wound response started with (1) local inhibition of active cytoplasmic streaming (unidirectional movements) due to inactivation, depolymerization, or mechanical displacement of the subcortical actin bundles. Accordingly, vesicles performed only oscillating motions and moved slowly with the same velocity and direction as passive endoplasmic flow. (2) Several minutes after wounding, vesicles started to perform random saltatory movements with frequently changing velocities, punctuated by oscillating motion and periods of immobility (docking) at the plasma membrane. Vesicle trajectories correlated with a fine-meshed actin network at the wound site. (3) Several hours after wounding, vesicles moved again unidirectionally along regenerated subcortical actin bundles. Spreading of vesicles (vesicle contents) was observed during wound wall formation, i.e., during the period of saltatory movements when vesicles had access to the plasma membrane. Dependent on the type of wound wall being secreted, three variants could be distinguished: (1) slow and continuous spreading over a time period of several seconds up to 30 min near the plasma membrane, (2) fast spreading within 80 ms inside an already formed wound wall, and/or (3) fast spreading at the plasma membrane. We conclude from our study that wounding-induced changes in vesicle dynamics are due to transient reorganization of the actin cytoskeleton from parallel bundles to a fine-meshed network. Furthermore, our results indicate that spreading of vesicle contents varies considerably with time and may be delayed by vesicle docking and/or discharge.