Hsp90 Mediates Membrane Deformation and Exosome Release.

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and membrane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deforming function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

PARL deficiency in mouse causes Complex III defects, coenzyme Q depletion, and Leigh-like syndrome.

The mitochondrial intramembrane rhomboid protease PARL has been implicated in diverse functions in vitro, but its physiological role in vivo remains unclear. Here we show that Parl ablation in mouse causes a necrotizing encephalomyelopathy similar to Leigh syndrome, a mitochondrial disease characterized by disrupted energy production. Mice with conditional PARL deficiency in the nervous system, but not in muscle, develop a similar phenotype as germline Parl KOs, demonstrating the vital role of PARL in neurological homeostasis. Genetic modification of two major PARL substrates, PINK1 and PGAM5, do not modify this severe neurological phenotype. Parl-/- brain mitochondria are affected by progressive ultrastructural changes and by defects in Complex III (CIII) activity, coenzyme Q (CoQ) biosynthesis, and mitochondrial calcium metabolism. PARL is necessary for the stable expression of TTC19, which is required for CIII activity, and of COQ4, which is essential in CoQ biosynthesis. Thus, PARL plays a previously overlooked constitutive role in the maintenance of the respiratory chain in the nervous system, and its deficiency causes progressive mitochondrial dysfunction and structural abnormalities leading to neuronal necrosis and Leigh-like syndrome.

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography.

BACKGROUND: Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. RESULTS: We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. CONCLUSIONS: Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals.

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.

TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport.

Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206-229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex.

BIG3 inhibits insulin granule biogenesis and insulin secretion.

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to ß-granin in ß-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Detection of insulin granule exocytosis by an electrophysiology method with high temporal resolution reveals enlarged insulin granule pool in BIG3-knockout mice.

We recently identified BIG3 as a negative regulator of insulin granule biogenesis and reported increased insulin secretion in BIG3-knockout (BKO) mice. To pinpoint the site of action for BIG3, we investigated whether BIG3 regulates quantal insulin granule exocytosis. We established an assay to detect insulin granule exocytosis by recording ATP-elicited currents at high temporal resolution by patch clamp. Similarly to insulin, ATP release was increased in BKO ß-cells. Although the frequency of insulin granule exocytosis was increased in BKO ß-cells, quantal size or release kinetics remained unchanged. Electron microscopy studies showed that the number of insulin granules was increased by >60% in BKO ß-cells. However, the number of morphologically docked granules was unaltered. The number of insulin granules having significant distances away from plasma membrane was greatly increased in BKO ß-cells. Thus, BIG3 negatively regulates insulin granule exocytosis by restricting insulin granule biogenesis without the release kinetics of individual granules at the final exocytotic steps being affected. Depletion of BIG3 leads to an enlarged releasable pool of insulin granules, which accounts for increased release frequency and consequently increased insulin secretion.

Cdk5-dependent rapid formation and stabilization of dendritic spines by corticotropin-releasing factor.

The neuropeptide corticotropin-releasing factor (CRF) exerts a pivotal role in modulating neuronal activity in the mammalian brain. The effects of CRF exhibit notable variations, depending on factors such as duration of exposure, concentration, and anatomical location. In the CA1 region of the hippocampus, the impact of CRF is dichotomous: chronic exposure to CRF impairs synapse formation and dendritic integrity, whereas brief exposure enhances synapse formation and plasticity. In the current study, we demonstrate long-term effects of acute CRF on the density and stability of mature mushroom spines ex vivo. We establish that both CRF receptors are present in this hippocampal region, and we pinpoint their precise subcellular localization within synapses by electron microscopy. Furthermore, both in vivo and ex vivo data collectively demonstrate that a transient surge of CRF in the CA1 activates the cyclin-dependent kinase 5 (Cdk5)-pathway. This activation leads to a notable augmentation in CRF-dependent spine formation. Overall, these data suggest that upon acute release of CRF in the CA1-SR synapse, both CRF-Rs can be activated and promote synaptic plasticity via activating different downstream signaling pathways, such as the Cdk5-pathway.