mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

Robust, fiducial-free drift correction for super-resolution imaging.

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

ATG9A protects the plasma membrane from programmed and incidental permeabilization.

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.

Endolysosomal Mesoporous Silica Nanoparticle Trafficking along Microtubular Highways.

This study examines intra- and intercellular trafficking of mesoporous silica nanoparticles along microtubular highways, with an emphasis on intercellular bridges connecting interphase and telophase cells. The study of nanoparticle trafficking within and between cells during all phases of the cell cycle is relevant to payload destination and dilution, and impacts delivery of therapeutic or diagnostic agents. Super-resolution stochastic optical reconstruction and sub-airy unit image acquisition, the latter combined with Huygens deconvolution microscopy, enable single nanoparticle and microtubule resolution. Combined structural and functional data provide enhanced details on biological processes, with an example of mitotic inheritance during cancer cell trivision.

MERIT, a cellular system coordinating lysosomal repair, removal and replacement.

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing ß-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau. ABBREVIATIONS: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16.

Galectin-3 Coordinates a Cellular System for Lysosomal Repair and Removal.

Endomembrane damage elicits homeostatic responses including ESCRT-dependent membrane repair and autophagic removal of damaged organelles. Previous studies have suggested that these systems may act separately. Here, we show that galectin-3 (Gal3), a ß-galactoside-binding cytosolic lectin, unifies and coordinates ESCRT and autophagy responses to lysosomal damage. Gal3 and its capacity to recognize damage-exposed glycans were required for efficient recruitment of the ESCRT component ALIX during lysosomal damage. Both Gal3 and ALIX were required for restoration of lysosomal function. Gal3 promoted interactions between ALIX and the downstream ESCRT-III effector CHMP4 during lysosomal repair. At later time points following lysosomal injury, Gal3 controlled autophagic responses. When this failed, as in Gal3 knockout cells, lysosomal replacement program took over through TFEB. Manifestations of this staged response, which includes membrane repair, removal, and replacement, were detected in model systems of lysosomal damage inflicted by proteopathic tau and during phagosome parasitism by Mycobacterium tuberculosis.

γ-secretase inhibitor DAPT mitigates cisplatin-induced acute kidney injury by suppressing Notch1 signaling.

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin-induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti-cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta-like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor reversed cisplatin-induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin-induced tubular injury and reduction in glomerular filtration rate. Real-time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin-treated mice which were abrogated by DAPT. Cisplatin-induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK-2). siRNA-mediated Dll1 knockdown and DAPT attenuated cisplatin-induced Notch1 cleavage and cytotoxicity in HK-2 cells. These data suggest that Dll1-mediated Notch1 signaling contributes to cisplatin-induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.

Sequential super-resolution imaging using DNA strand displacement.

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with ß-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

Dysferlin forms a dimer mediated by the C2 domains and the transmembrane domain in vitro and in living cells.

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.