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Review
. 2022 Sep 2:13:983152.
doi: 10.3389/fendo.2022.983152. eCollection 2022.

The changing view of insulin granule mobility: From conveyor belt to signaling hub

Bastian Gaus  1 , Dennis Brüning  1 , Sofie Groß  1 , Michael Müller  2 , Ingo Rustenbeck  1
Affiliations
Review

The changing view of insulin granule mobility: From conveyor belt to signaling hub

Bastian Gaus et al. Front Endocrinol (Lausanne). .

Abstract

Before the advent of TIRF microscopy the fate of the insulin granule prior to secretion was deduced from biochemical investigations, electron microscopy and electrophysiological measurements. Since Calcium-triggered granule fusion is indisputably necessary to release insulin into the extracellular space, much effort was directed to the measure this event at the single granule level. This has also been the major application of the TIRF microscopy of the pancreatic beta cell when it became available about 20 years ago. To better understand the metabolic modulation of secretion, we were interested to characterize the entirety of the insulin granules which are localized in the vicinity of the plasma membrane to identify the characteristics which predispose to fusion. In this review we concentrate on how the description of granule mobility in the submembrane space has evolved as a result of progress in methodology. The granules are in a state of constant turnover with widely different periods of residence in this space. While granule fusion is associated +with prolonged residence and decreased lateral mobility, these characteristics may not only result from binding to the plasma membrane but also from binding to the cortical actin web, which is present in the immediate submembrane space. While granule age as such affects granule mobility and fusion probability, the preceding functional states of the beta cell leave their mark on these parameters, too. In summary, the submembrane granules form a highly dynamic heterogeneous population and contribute to the metabolic memory of the beta cells.

Keywords: TIRF microscopy; actin; calcium; insulin granules; insulin secretion; pancreatic islet.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Growing complexity of the relation between granule mobility and fusion competence. The electrophysiological investigations (left part of the sketch) led to the definition of a readily releasable pool of docked and primed granules (highlighted by red rims), the emptying of which caused the nadir between the first and the second phase of insulin secretion. The slow refilling from a distant reserve pool was considered as the cause of the slower pace of secretion during the second phase. The initial TIRF studies (second from left) considered the predocked granules as the correlate of the readily releasable pool and noted an increasingly relevant contribution of newcomer granules (highlighted by yellow rims) with time. Later (second from right), a predominant contribution of restless newcomer granules to the first phase was described. The lower fusion rates during prolonged stimulation were ascribed to the passage through the cortical actin layer. The rightmost part of the sketch combines two sets of observations. The cortical actin layer, which extends to the immediate vicinity of the plasma membrane is not just an obstacle, but a near-membrane storage site of granules. There is a constant exchange of granules which arrive at the submembrane space and leave again after different periods of residence, mostly within one second. Fusion is possible after varying periods of residence.
Figure 2
Figure 2
Colocalization of insulin granules and actin in the submembrane space of MIN6 cells. The insulin granules of MIN6-cells (p20 - p30) were labeled by transient transfection with hIns-EGFP and mTagRFP-T-Lifeact-7. The fluorescence emission < 560 nm (upper image) gives the conventional TIRFM image of green fluorescent granules in the submembrane space (calculated decay constant 85 nm). The fluorescence emission > 560 nm shows the presence of red-labelled actin (middle image) together with insulin granules (overlay, lower image) in the same space. The coexistence suggests that the granule mobility may reflect, at least in part, interactions with the cortical actin.
Figure 3
Figure 3
3D view of insulin granules and actin in primary beta cells. The insulin granules of primary beta cells were labeled by adenoviral transfection with hIns-EGFP (green) and mTagRFP-T-Lifeact-7 (orange). The fluorescence was excited by spinning disk CLSM (50 µm disk, 2.8fold magnification by SORA attachment, objective Nikon SR HP Apo TIRF 100x, N.A. 1.49). The z-stack was generated from 61 levels at 200 nm steps. The lower images show the same beta cell as above, but viewed from an oblique angle. The right images show the same cell as the left images after 10 min incubation in the presence of latrunculin. Note the decrease of actin by latrunculin all around the beta cell.
Figure 4
Figure 4
Simulation of insulin secretion as function of granule mobility. Consequences of varying the simulation parameters. The consequences were tested by halving the parameter values one by one while fixing all other parameters. When this results in the general increase of diffuse granule movement over directional movement, e.g. by lower chord lengths in the actin network, secretion is markedly reduced, even in the stationary phase (green line). Halving the parameter value describing the transition from diffuse movement into directional movement, such as generated by the increase of the cytosolic Ca2+ concentration, results in a similar but less pronounced decrease (red line). When the effect of halving results in the prolonged presence of the granules at the membrane prior to release, the peak value of secretion is retarded, but the stationary phase remains unchanged (yellow line). Adapted from .
Figure 5
Figure 5
Presence of insulin granules and mitochondria in the submembrane space of MIN6 cells. The insulin granules of MIN6 cells were transiently labelled with hIns-EGFP (green) and mitochondria by loading with TMRE. The image acquisition of the z-stack was performed as in Figure 3 . The left image shows the cell from the bottom - attachment to the cover slip, the right image shows the lateral view to demonstrate the high concentration of mitochondria in the immediate vicinity of the submembrane space.
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