3D FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse beta cells

Abstract

Microtubules play a major role in intracellular trafficking of cargo vesicles in endocrine cells and detailed knowledge of the microtubule network organization and its relation to other cell constituents is crucial for understanding primary cell function. However, their role in insulin transport and secretion is currently under debate. Here, we use Fib-Sem to image insulin secreting pancreatic islet beta cells in their entirety at an unprecedented resolution. We reconstruct all mitochondria, Golgi apparati, centrioles, insulin secretory granules and microtubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that microtubules form non-radial networks that are predominantly not connected to either centrioles or endomembranes. Moreover, microtubule number and length, but not microtubule density, vary with glucose stimulation. Furthermore, we show that insulin secretory granules are enriched near the plasma membrane where they associate with microtubules. In summary, we provide the first three-dimensional reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus supportive role in insulin secretion.

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

“…In this way, secretory sites in β-cells are reminiscent of LL5βand CLASP-containing podosome-like structures ("synaptic podosomes"), which provide a route for microtubule-based delivery and accumulation of acetylcholine receptors at neuromuscular junctions in skeletal muscle cells (Basu et al, 2015, Kishi et al, 2005, Proszynski et al, 2009, Proszynski et al, 2013, Schmidt et al, 2012. However, although microtubules are in tight contact with the secretion complexes and insulin granules (Muller et al, 2021, Yuan et al, 2015, it is unlikely that the loss of LL5β perturbs the rapid phase of insulin exocytosis by reducing microtubule density. First, this secretion phase depends on insulin granules that are already docked at the cortex and do not need to be transported along microtubules.…”

An open-access volume electron microscopy atlas of whole cells and tissues

“…The seamless imaging results in precisely aligned stacks furthermore facilitating automated 3D segmentation. Specifically, from these FIB-SEM datasets has emerged a comprehensive 3D representation of microtubule networks and how they interact with other organelles 16 . We could show that beta cell microtubules are arranged into non-radial networks which for the most part do not originate either from centrioles or endomembranes.…”

“…For example, the datasets of mouse pancreatic islets (Fig. 4a–4f) unveil a comprehensive 3D representation of the microtubule network and how it interacts with other organelles, as well as microtubule remodeling as a result of glucose stimulation 16 . Likewise, the data of HeLa and U2-OS nuclei were mined to identify and characterize chromatin domains 19 .…”

The use of soft X-ray tomography to explore mitochondrial structure and function

“…SXT generates a 3D reconstructed volume of an entire cell within 10 min. Other 3D imaging methods require time-consuming serial sectioning and may allow for comparison of only a few cells [ 28 , 29 ] while SXT has been used to compare tens or hundreds of cells, depending on the cell type. For example, the structural organization of 60 INS-1E cells (a rat insulinoma cell line) under different conditions has been compared [ 16 ].…”

“…May also require plastic embedding or chemical fixation. [ 28 , 29 , 56 , 57 ] Cryo-electron tomography (thin periphery of cell, thin lamella of interior of cell, single-cell multiple distinct tomogram acquisitions) 2–5 nm in 180–250 nm thick samples Provides high resolution details on 3D cellular organization within windows of the cell. Allows for in-situ structural biology.…”