Project description:Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Conventional protein delivery techniques fail to address this fundamental problem in that protein cargos are predominantly delivered inside cells via endocytosis, a remarkably effective cell defense mechanism developed by Mother Nature to prevent intact biomolecules from entering the cytoplasm. Here, we report a unique concept, noncovalent cholesterol tagging, enabling virtually any compact proteins to permeate through the cell membrane, completely bypassing endocytosis. This simple plug-and-play platform greatly expands the biological target space and has the potential to transform basic biology studies and drug discovery.
Project description:The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9. APEX2-catalyzed gold/silver precipitation markedly improved EM clarity and confirmed an apparent intra-nuclear presence of Sig1R. However, serial sectioning combined with APEX2-enhanced EM revealed that Sig1R actually resided in the nucleoplasmic reticulum (NR), a specialized nuclear compartment formed via NE invagination into the nucleoplasm. NR cross-sections also indicated Sig1R in ring-shaped NR membranes. Thus, this study distinguishes Sig1R in the NR which could otherwise appear localized in the nucleoplasm if detected with low-resolution methods. Our finding is important for uncovering potential Sig1R regulations in the nucleus.
Project description:Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.
Project description:BackgroundIn eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.ResultsHere, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport.ConclusionAltogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.
Project description:Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.
Project description:Subcellular localization of the transcription factor Prospero is dynamic. For example, the protein is cytoplasmic in neuroblasts, nuclear in sheath cells, and degraded in newly formed neurons. The carboxy terminus of Prospero, including the homeodomain and Prospero domain, plays roles in regulating these changes. The homeodomain has two distinct subdomains, which exclude proteins from the nucleus, while the intact homeo/Prospero domain masks this effect. One subdomain is an Exportin-dependent nuclear export signal requiring three conserved hydrophobic residues, which models onto helix 1. Another, including helices 2 and 3, requires proteasome activity to degrade nuclear protein. Finally, the Prospero domain is missing in pros(I13) embryos, thus unmasking nuclear exclusion, resulting in constitutively cytoplasmic protein. Multiple processes direct Prospero regulation of cell fate in embryonic nervous system development.
Project description:Despite substantial recent progress in network neuroscience, the impact of stroke on the distinct features of reorganizing neuronal networks during recovery has not been defined. Using a functional connections-based approach through 2-photon in vivo calcium imaging at the level of single neurons, we demonstrate for the first time the functional connectivity maps during motion and nonmotion states, connection length distribution in functional connectome maps and a pattern of high clustering in motor and premotor cortical networks that is disturbed in stroke and reconstitutes partially in recovery. Stroke disrupts the network topology of connected inhibitory and excitatory neurons with distinct patterns in these 2 cell types and in different cortical areas. These data indicate that premotor cortex displays a distinguished neuron-specific recovery profile after stroke.
Project description:Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.
Project description:The major constituent of the eukaryotic ER protein-translocation channel (Sec61p in yeast, Sec61α in higher eukaryotes) shows a high degree of evolutionary conservation from yeast to humans. The vast majority of eukaryotic species have a conserved di-tyrosine in the 4th ER luminal loop. Previously, we discovered through a screen of ethylnitrosourea- (ENU-) mutagenized mice that substitution of the latter of these tyrosines with histidine (Y344H) of the murine Sec61α protein results in diabetes and hepatic steatosis in mice that is a result of ER stress. To further characterize the mechanism behind ER stress in these mice we made the homologous mutation in yeast Sec61p (Y345H). We found that this mutation increased sensitivity of yeast to ER stressing agents and to reduction of Inositol Requiring Enzyme 1 (IRE1) activity. Furthermore, we found that, while this mutation did not affect translocation, it did delay degradation of the model ER-associated degradation (ERAD) substrate CPY(∗). Replacing both ER luminal tyrosines with alanines resulted in a destabilization of the Sec61 protein that was rescued by over expression of Sss1p. This double mutant still lacked a noticeable translocation defect after stabilization by Sss1p, but exhibited a similar defect in CPY(∗) degradation.
Project description:Endoplasmic reticulum quality control is crucial for maintaining cellular homeostasis and adapting to stress conditions. Although several ER-phagy receptors have been identified, the collaboration between cytosolic and ER-resident factors in ER fragmentation and ER-phagy regulation remains unclear. Here, we perform a phenotype-based gain-of-function screen and identify a cytosolic protein, FKBPL, functioning as an ER-phagy regulator. Overexpression of FKBPL triggers ER fragmentation and ER-phagy. FKBPL has multiple protein binding domains, can self-associate and might act as a scaffold connecting CKAP4 and LC3/GABARAPs. CKAP4 serves as a bridge between FKBPL and ER-phagy cargo. ER-phagy-inducing conditions increase FKBPL-CKAP4 interaction followed by FKBPL oligomerization at the ER, leading to ER-phagy. In addition, FKBPL-CKAP4 deficiency leads to Golgi disassembly and lysosome impairment, and an increase in ER-derived secretory vesicles and enhances cytosolic protein secretion via microvesicle shedding. Taken together, FKBPL with the aid of CKAP4 induces ER fragmentation and ER-phagy, and FKBPL-CKAP4 deficiency facilitates protein secretion.