Journal of Cell Biology最新文献

Insulin granule fusion in pancreatic β cells localizes to where they contact the ECM of the islet capillaries. The mechanism(s) underpinning localization are unclear. Using glucose or high K+ stimulation or the global uncaging of Ca2+, we show granule fusion consistently focused to the β cell-ECM interface, suggesting a specific localization mechanism. We tested for the involvement of liprin-α1, a scaffold protein enriched at the β cell-ECM interface. Liprin-α1 knockdown did not affect high K+-stimulated insulin secretion but did impair localization of exocytosis. Liprin-α1 knockdown impaired glucose-induced insulin secretion with evidence that the C-terminal of liprin-α1 positions liprin-α1 in clusters at the β cell-ECM interface. Liprin-α1 cluster size and number are regulated by glucose, and exocytosis is spatially coupled with the clusters. Immunoprecipitation and mass spectrometry characterized a liprin-α1 interactome, including β2-syntrophin, an insulin granule-linked protein. We conclude that liprin-α1 is part of a complex that is regulated by glucose and locally targets insulin granules to the β cell-ECM interface.

TFEB, a master regulator of autophagy and lysosomal biogenesis, is activated by several cellular stresses including lysosomal damage, but its underlying mechanism is unclear. TFEB activation during lysosomal damage depends on the ATG conjugation system, which mediates lipidation of ATG8 proteins. Here, we newly identify ATG conjugation-independent TFEB regulation that precedes ATG conjugation-dependent regulation, designated Modes I and II, respectively. We reveal unique regulators of TFEB in each mode: APEX1 in Mode I and CCT7 and/or TRIP6 in Mode II. APEX1 interacts with TFEB independently of the ATG conjugation system, and is required for TFEB stability, while both CCT7 and TRIP6 accumulate on lysosomes during lysosomal damage, and interact with TFEB mainly in ATG conjugation system-deficient cells, presumably blocking TFEB activation. TFEB activation by several other stresses also involves either Mode I or Mode II. Our results pave the way for a unified understanding of TFEB regulatory mechanisms from the perspective of the ATG conjugation system under a variety of cellular stresses.

The secretion of extracellular matrix (ECM) proteins is vital to the maintenance of tissue health. One major control point of this process is the Golgi apparatus, whose dysfunction causes numerous connective tissue disorders. We therefore sought to investigate the role of two Golgi organizing proteins, GMAP210 and Golgin-160, in ECM secretion. CRISPR knockout of either protein had distinct impacts on Golgi organization, with Golgin-160 knockout causing Golgi fragmentation and vesicle accumulation, and GMAP210 loss leading to cisternal fragmentation, dilation, and the accumulation of tubulovesicular structures. Both golgins were required for fibrillar collagen organization and glycosaminoglycan synthesis suggesting nonredundant functions in these processes. Furthermore, proteomics analysis revealed both shared and golgin-specific changes in the secretion of ECM proteins. We therefore propose that golgins are collectively required to create the correct physical-chemical space to support efficient ECM protein secretion, modification, and assembly. This is the first time that Golgin-160 has been shown to be required for ECM secretion.

Microtubules are cytoskeletal filaments that provide structural support for numerous cellular processes. Despite their high rigidity, microtubules can be dramatically bent in cells, and it is unknown how much force a microtubule can withstand before breaking. We find that the kinesin-3 motor KIF1C forms condensates that entangle and break neighboring microtubules. Combining computational simulations and experiments, we show that microtubule breakage is an emergent property that is dependent on a highly processive kinesin motor domain, the cluster properties, cytoplasmic viscosity, and microtubule anchors. We estimate a rupture force for microtubules in cells that is lower than previous estimates based on in vitro studies with taxol-stabilized microtubules. The absence of microtubule breakage under physiological conditions suggests that mechanisms exist to protect microtubule integrity, which may inform about physical constraints on the evolution of motor proteins. We suggest that release of either the motor-cargo or motor-microtubule interaction prevents the accumulation of mechanical stress upon the engagement of multi-motor clusters with microtubules.

Membrane contact sites (MCSs) establish organelle interactomes in cells to enable communication and exchange of materials. Volume EM (vEM) is ideally suited for MCS analyses, but semantic segmentation of large vEM datasets remains challenging. Recent adoption of artificial intelligence (AI) for segmentation has greatly enhanced our analysis capabilities. However, we show that organelle boundaries, which are important for defining MCS, are the least confident predictions made by AI. We outline a segmentation strategy termed AI-directed voxel extraction (AIVE), which refines segmentation results and boundary predictions derived from any AI-based method by combining those results with electron signal values. We demonstrate the precision conferred by AIVE by applying it to the quantitative analysis of organelle interactomes from multiple FIB-SEM datasets. Through AIVE, we discover a previously unknown category of mitochondrial contact that we term the mitochondrial intrusion. We hypothesize that intrusions serve as anchors that stabilize MCS and promote organelle communication.