Restriction of secretory granule motion near the plasma membrane of chromaffin cells.
We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.
Johns
L M
LM
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Levitan
E S
ES
Shelden
E A
EA
Holz
R W
RW
Axelrod
D
D
D000199:Actins / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D019274:Botulinum Toxins, Type A; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D019439:Chromaffin Cells / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D004058:Diffusion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D008856:Microscopy, Fluorescence / Q000379:methods; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D013844:Thiazoles / Q000378:metabolism; D053778:Thiazolidines; D013997:Time Factors
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D013844:Thiazoles; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; D009320:Atrial Natriuretic Factor; D019274:Botulinum Toxins, Type A; C037067:latrunculin A