Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs’ three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non-epithelial spaces (stroma) of many organs (see www.telocytes.com). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB-SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB-SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB-SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.

Key Points• WPBs stay connected to the Golgi apparatus until vesicle formation is completed.• During biogenesis at the Golgi, WPBs increase in size through the addition of nontubular VWF.Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs. (Blood. 2015;125(22):3509-3516) IntroductionRapid secretion of the endothelial storage organelles, the WeibelPalade bodies (WPBs), 1 is fundamental for hemostasis. WPBs contain the hemostatic glycoprotein von Willebrand factor (VWF), which recruits platelets to sites of injury to arrest bleeding.2 Within WPBs, VWF is packed into helical tubules that give the organelle an elongated shape with a length of 1 to 5 mm and a width of 100 to 300 nm. 1,[3][4][5] The formation of WPBs is dependent on VWF and also occurs on VWF expression in nonendothelial cells. 6,7 VWF is synthesized in the endoplasmic reticulum as a pre-proprotein consisting of a signal peptide, a propeptide, and mature VWF.2 On removal of the signal peptide, VWF dimers are formed that are transported to the Golgi apparatus. At the Golgi, the propeptide is cleaved from mature VWF to guide multimerization and tubule formation. 3,8,9 VWF tubule formation is crucial for the development of mature, densely packed elongated WPBs. Mutations in the VWF gene, as found in patients with the bleeding disorder von Willebrand disease, were shown to result in altered WPB morphology. 2,[10][11][12] In vitro studies on VWF tubule formation demonstrated that the core of the VWF tubules is formed by the propeptide and the N-terminal D9 and D3 assembly of mature VWF.3 However, it is still poorly understood how the formation of the VWF tubules is related to WPB biogenesis.Electron microscopy studies have revealed several stages in the WPB formation pro...

We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. Conclusions: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions. K E Y W O R D S3D-confocal fluorescence imaging, 3D-scanning electron microscopy (SEM), cellular fibronectin EDA domain, connexin 43 (Cx43), Interstitial Cells of Cajal (ICC), telocyte, wheat germ agglutinin (WGA) staining Abbreviations: aSMCA, alpha smooth muscle cell actin; bIC, branched interstitial cell; BL, basal lamina; BM, (urothelial) basal membrane; fIC, fibroblast type interstitial cell; FIB-SEM, focused ion beam scanning electron microscopy; GJ, gap junction; mIC, myoid interstitial cell; rER, rough endoplasmic reticulum; sER, smooth endoplasmic reticulum; UC, urothelial cell; ULP, upper lamina propria.