scholarly journals Restriction of Secretory Granule Motion near the Plasma Membrane of Chromaffin Cells

2001 ◽  
Vol 153 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Laura M. Johns ◽  
Edwin S. Levitan ◽  
Eric A. Shelden ◽  
Ronald W. Holz ◽  
Daniel Axelrod
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Chromaffin Cells ◽  
Botulinum Toxin A ◽  
Secretory Granules ◽  
Internal Reflection ◽  
Snare Proteins ◽  
Bovine Chromaffin Cells ◽  
Heterogeneous Matrix ◽  
Granule Motion

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 ∼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.

scholarly journals Motion Matters: Secretory Granule Motion Adjacent to the Plasma Membrane and Exocytosis

2006 ◽  
Vol 17 (5) ◽  
pp. 2424-2438 ◽  
Author(s):  
Miriam W. Allersma ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Chromaffin Cells ◽  
Actin Dynamics ◽  
Permeabilized Cells ◽  
Calcium Dependent ◽  
Individual Granule ◽  
Granule Motion

Total internal reflection fluorescence microscopy was used to monitor changes in individual granule motions related to the secretory response in chromaffin cells. Because the motions of granules are very small (tens of nanometers), instrumental noise in the quantitation of granule motion was taken into account. ATP and Ca2+, both of which prime secretion before fusion, also affect granule motion. Removal of ATP in permeabilized cells causes average granule motion to decrease. Nicotinic stimulation causes a calcium-dependent increase in average granule motion. This effect is more pronounced for granules that undergo exocytosis than for those that do not. Fusion is not preceded by a reduction in mobility. Granules sometimes move 100 nm or more up to and within a tenth of a second before fusion. Thus, the jittering motion of granules adjacent to the plasma membrane is regulated by factors that regulate secretion and may play a role in secretion. Motion continues until shortly before fusion, suggesting that interaction of granule and plasma membrane proteins is transient. Disruption of actin dynamics did not significantly alter granule motion.

Submembrane Events In Triggerable Cells Studied by Total Internal Reflection Fluorescence Microscopy.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1052-1053
Author(s):  
D. Axelrod ◽  
L.M. Johns ◽  
E.S. Levitan ◽  
G.M. Omann ◽  
R.W. Holz
Keyword(s):  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Cell Substrate ◽  
Before And After ◽  
Cell Medium ◽  
Very High

We have studied the dynamics of certain key processes near the plasma membrane inside two types of chemically-triggerable living cells using total internal reflection fluorescence microscopy (TIRFM). In TIRFM, a laser beam is incident upon the cell/glass-substrate interface from the glass side at an angle greater than the critical angle for total internal reflection. This creates an exponentially decaying evanescent field in the cell medium (with a characteristic depth of > 100 nm) capable of exciting fluorescence selectively from the membrane-proximal regions at cell/substrate contacts. Various ways of setting up the optics for such a system are discussed, involving the use of either prisms or very high aperture objectives.In one application of TIRFM, the motion of adrenalin-containing secretory granules in the immediate submembrane region of chromaffin cells is examined before and after chemical stimulation that causes the granules to release their contents to the cell exterior.

scholarly journals Visualization of Regulated Exocytosis with a Granule-Membrane Probe Using Total Internal Reflection Microscopy

2004 ◽  
Vol 15 (10) ◽  
pp. 4658-4668 ◽  
Author(s):  
Miriam W. Allersma ◽  
Li Wang ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Evanescent Field ◽  
Internal Reflection ◽  
Spherical Granule ◽  
Granule Membrane ◽  
Green Fluorescent

Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In ∼90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of ∼200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, ∼20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, ∼10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

2006 ◽  
Vol 291 (1) ◽  
pp. G146-G155 ◽  
Author(s):  
Jong Hak Won ◽  
David I. Yule
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Acinar Cells ◽  
Internal Reflection ◽  
Pancreatic Acinar Cells ◽  
Wide Field ◽  
Exocrine Cells ◽  
Parotid Acinar Cells ◽  
Signaling Dynamics ◽  
Total Internal Reflection Microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

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