Brain Cell Biology

, Volume 35, Issue 1, pp 57–73

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

  • Guy Malkinson
  • Zohar M. Fridman
  • Dotan Kamber
  • Ada Dormann
  • Eli Shapira
  • Micha E. Spira
Research Article

Abstract

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane “sleeve”, split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities’ organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as “ready to go” prefabricated presynaptic terminals. We suggest that the varicosities’ motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse.

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Supplementary material

Movie 1

(Fig. 1A). The motile behavior of VRs formed by a cultured metacerebral neuron. Frames taken every 4 minutes for a period of 40 minutes. Display rate: 6 frames per second (f.p.s).

11068_2006_9007_MOESEM2_ESM.avi (415 kb)
Movie 2(Fig. 1C). The VR on the left propagates along the neurite and merges with a second VR on its path, forming a single, larger VR. Frames taken every 5 minutes for a period of 45 minutes. Display rate: 4 f.p.s.
11068_2006_9007_MOESEM3_ESM.avi (30 kb)
Movie 3(Fig. 1E). A wobbling VR. Frames taken every 60 seconds. for a period of 45 minutes. Display rate: 4 f.p.s.
11068_2006_9007_MOESEM4_ESM.avi (663 kb)
Movie 4(Fig 4A). A translocation of a giant cluster of RH237 labeled organelles enwrapped by actin from the VR on the left to the one on the right. Upper rowtransmitted image. Red- RH237 labeled organelles. Green- EGFP-actin. Lower rowmerged image. Frames taken every 60 seconds. Frame rate: 5fps.
Movie 5

A translocation of a VR containing RH237 labeled organelles enwrapped by actin. Left- transmitted image. Red- RH237 labeled organelles. Green- EGFP-actin. rightmerged image. Frames taken every 60 seconds. Frame rate: 5 f.p.s.

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Movie 6A concerted movement of mitochondria aggregates labeled by RPAC and the VR that contains them. Frames taken every 45 seconds. Frame rate: 6 f.p.s.
11068_2006_9007_MOESEM7_ESM.avi (409 kb)
Movie 7(Fig. 5C). A cultured neuron expressing PA- SNAP25. A single VR was activated. Following activation the VR split, passing a part of its vesicles content to the newly formed VR. Left- transmitted image. Right- PA-SNAP25. Frames taken every 30 seconds. Frame rate: 4 f.p.s.
Movie 8

(Fig. 6). A cultured neuron expressing kaede-actin. A single VR was photo switched. Following the switch, the VR split, contributing a part of its actin to the new formed VR. Left- transmitted image. Green -actin kaede before switch. Red- actin kaede after switch. Right- merged image. Frames taken every 180 seconds. Frame rate: 4 f.p.s.

11068_2006_9007_MOESEM9_ESM.avi (687 kb)
Movie 9A motile VR retains its activity-dependent calcium-influx capabilities, evident by translocation of EGFP-DOC2B at different locations. Frames taken every 60 seconds. Frame rate: 6 f.p.s.
11068_2006_9007_MOESEM10_ESM.avi (794 kb)
Movie 10A motile VR retains its activity-dependent exocytosis, evident by a rise in the synpH signal at the two locations. Frames taken every 90 seconds. Frame rate: 5 f.p.s.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Guy Malkinson
    • 1
  • Zohar M. Fridman
    • 1
  • Dotan Kamber
    • 1
  • Ada Dormann
    • 1
  • Eli Shapira
    • 1
  • Micha E. Spira
    • 1
  1. 1.Department of Neurobiology, Institute of Life SciencesThe Hebrew University of JerusalemJerusalemIsrael

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