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

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.

Liquid condensate is a common state of proteins and polypeptides at the regime of high intermolecular interactions

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological mechanism for sequestering macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides at their high intermolecular interaction regime. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study using molecular crowder polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase space and conditions. Using a single component and combinations of protein multicomponent (co-LLPS) systems, we establish that a variety of intermolecular interactions can drive proteins/polypeptides LLPS.

Temperature-dependent Reentrant Phase Transition of RNA-polycation Mixtures

Liquid-liquid phase separation (LLPS) of multivalent biopolymers is a ubiquitous process in biological systems and is of importance in bio-mimetic soft matter design. The phase behavior of biomolecules, such as...

Valosin-containing protein regulates the stability of amyotrophic lateral sclerosis-causing fused in sarcoma granules in cells by changing ATP concentrations inside the granules

Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) to form granules in cells, leading to pathogenic aggregations that cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Proteomics analysis revealed that FUS granules contain valosin-containing protein (VCP), a member of the AAA family ATPase. Confocal microscopy images showed that VCP co-localized in the FUS granules in cells. This study demonstrates that VCP in granules has a two-faced role in FUS granulation: VCP stabilizes de novo FUS granules, while VCP present in the granules for extended periods dissolves them. This VCP function relies on its ATPase activity to consume ATP in granules. VCP stabilizes de novo FUS by reducing intragranular ATP concentrations to a range below the cytosolic concentration. VCP continually consumes ATP during its stay in the granules, which eventually lowers ATP concentrations to a range that destabilizes the granules. VCP, therefore, acts as a timer to limit the residence of FUS granules in cells and thereby prohibits the FUS fibrillization that occurs in persistent granules. VCP ATPase activity plays a role in FUS granule turnover.

Liquid-Liquid Phase Separation Phenomenon on Protein Sorting Within Chloroplasts

In higher plants, chloroplasts are vital organelles possessing highly complex compartmentalization. As most chloroplast-located proteins are encoded in the nucleus and synthesized in the cytosol, the correct sorting of these proteins to appropriate compartments is critical for the proper functions of chloroplasts as well as plant survival. Nuclear-encoded chloroplast proteins are imported into stroma and further sorted to distinct compartments via different pathways. The proteins predicted to be sorted to the thylakoid lumen by the chloroplast twin arginine transport (cpTAT) pathway are shown to be facilitated by STT1/2 driven liquid-liquid phase separation (LLPS). Liquid-liquid phase separation is a novel mechanism to facilitate the formation of membrane-less sub-cellular compartments and accelerate biochemical reactions temporally and spatially. In this review, we introduce the sorting mechanisms within chloroplasts, and briefly summarize the properties and significance of LLPS, with an emphasis on the novel function of LLPS in the sorting of cpTAT substrate proteins. We conclude with perspectives for the future research on chloroplast protein sorting and targeting mechanisms.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules

Insulin is a key regulator of human metabolism, and its dysfunction leads to diseases such as type 2 diabetes. It remains unknown how proinsulin is targeted from the trans-Golgi network (TGN) to secretory storage granules as no cargo receptor has been identified. Chromogranin proteins (CGs) are central regulators of granule biosynthesis, and it was proposed that their aggregation is critical for this process. However, the molecular mechanism by which these molecules facilitate sorting at the TGN is poorly understood. Here, we show that CGs undergo liquid-liquid phase separation (LLPS) at low pH independently of divalent cations, such as calcium. Liquid CG condensates, but not aggregates, recruit and sort proinsulin and other granule destined cargo molecules towards secretory granules. Cargo selectivity is independent of sequence or structural elements but is based on the size and concentration of the client molecules at the TGN. Finally, electrostatic interactions and the N-terminal intrinsically disordered domain of chromogranin B facilitate LLPS and are critical for granule formation. We propose that phase-separated CGs act as a cargo sponge within the TGN lumen, gathering soluble client proteins into the condensate independently of specific sequence or structural elements, facilitating receptor-independent sorting. These findings challenge the canonical TGN sorting models and provide insights into granule biosynthesis in insulin-secreting beta cells.