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Pulses of RhoA signaling stimulate actin polymerization and flow in protrusions to drive collective cell migration

Qian, Weiyi; Yamaguchi, Naoya; Lis, Patrycja; Cammer, Michael; Knaut, Holger
In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin-II-dependent actin flow and protrusion retraction at the base of the protrusions and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other contexts.
PMID: 38096821
ISSN: 1879-0445
CID: 5588892

Computational Prediction of Coiled-Coil Protein Gelation Dynamics and Structure

Britton, Dustin; Christians, Luc F; Liu, Chengliang; Legocki, Jakub; Xiao, Yingxin; Meleties, Michael; Yang, Lin; Cammer, Michael; Jia, Sihan; Zhang, Zihan; Mahmoudinobar, Farbod; Kowalski, Zuzanna; Renfrew, P Douglas; Bonneau, Richard; Pochan, Darrin J; Pak, Alexander J; Montclare, Jin Kim
Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled-coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled-coil hydrogel design. Using Rosetta energy scores and Poisson-Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled-coil proteins while simultaneously exploring five new coiled-coil protein hydrogel sequences. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled-coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled-coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled-coil fiber formation.
PMID: 38110299
ISSN: 1526-4602
CID: 5611722

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen Encephalitozoon intestinalis

Antao, Noelle V; Lam, Cherry; Davydov, Ari; Riggi, Margot; Sall, Joseph; Petzold, Christopher; Liang, Feng-Xia; Iwasa, Janet H; Ekiert, Damian C; Bhabha, Gira
Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.
PMID: 37996434
ISSN: 2041-1723
CID: 5608812

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

Zhao, Yu; Coelho, Camila; Hughes, Amanda L; Lazar-Stefanita, Luciana; Yang, Sandy; Brooks, Aaron N; Walker, Roy S K; Zhang, Weimin; Lauer, Stephanie; Hernandez, Cindy; Cai, Jitong; Mitchell, Leslie A; Agmon, Neta; Shen, Yue; Sall, Joseph; Fanfani, Viola; Jalan, Anavi; Rivera, Jordan; Liang, Feng-Xia; Bader, Joel S; Stracquadanio, Giovanni; Steinmetz, Lars M; Cai, Yizhi; Boeke, Jef D
The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASer
PMID: 37944511
ISSN: 1097-4172
CID: 5590882

The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus replication

Li, Jie; Gui, Qi; Liang, Feng-Xia; Sall, Joseph; Zhang, Qingyue; Duan, Yatong; Dhabaria, Avantika; Askenazi, Manor; Ueberheide, Beatrix; Stapleford, Kenneth A; Pagano, Michele
Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.
PMID: 37768083
ISSN: 1098-5514
CID: 5614142

A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion

Li, Jie; Krause, Gregory J; Gui, Qi; Kaushik, Susmita; Rona, Gergely; Zhang, Qingyue; Liang, Feng-Xia; Dhabaria, Avantika; Anerillas, Carlos; Martindale, Jennifer L; Vasilyev, Nikita; Askenazi, Manor; Ueberheide, Beatrix; Nudler, Evgeny; Gorospe, Myriam; Cuervo, Ana Maria; Pagano, Michele
Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
PMID: 37831778
ISSN: 2375-2548
CID: 5604232

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching

Robertson, Amicha; Sall, Joseph; Venzon, Mericien; Olivas, Janet J; Zheng, Xuhui; Cammer, Michael; Antao, Noelle; Zhou, Chunyi; Devlin, Joseph C; Saes Thur, Rafaela; Bethony, Jeffrey; Nejsum, Peter; Shopsin, Bo; Torres, Victor J; Liang, Feng-Xia; Cadwell, Ken
The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.
PMID: 37738244
ISSN: 1553-7374
CID: 5627842

A membrane-associated MHC-I inhibitory axis for cancer immune evasion

Chen, Xufeng; Lu, Qiao; Zhou, Hua; Liu, Jia; Nadorp, Bettina; Lasry, Audrey; Sun, Zhengxi; Lai, Baoling; Rona, Gergely; Zhang, Jiangyan; Cammer, Michael; Wang, Kun; Al-Santli, Wafa; Ciantra, Zoe; Guo, Qianjin; You, Jia; Sengupta, Debrup; Boukhris, Ahmad; Zhang, Hongbing; Liu, Cheng; Cresswell, Peter; Dahia, Patricia L M; Pagano, Michele; Aifantis, Iannis; Wang, Jun
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
PMID: 37557169
ISSN: 1097-4172
CID: 5602312

Mitophagy promotes resistance to BH3 mimetics in acute myeloid leukemia

Glytsou, Christina; Chen, Xufeng; Zacharioudakis, Emmanouil; Al-Santli, Wafa; Zhou, Hua; Nadorp, Bettina; Lee, Soobeom; Lasry, Audrey; Sun, Zhengxi; Papaioannou, Dimitrios; Cammer, Michael; Wang, Kun; Zal, Tomasz; Zal, Malgorzata Anna; Carter, Bing Z; Ishizawa, Jo; Tibes, Raoul; Tsirigos, Aristotelis; Andreeff, Michael; Gavathiotis, Evripidis; Aifantis, Iannis
BH3-mimetics are used as an efficient strategy to induce cell death in several blood malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML. Moreover, MCL-1 or dual BCL-2/BCL-xL antagonists are under investigation. Yet, resistance to single or combinatorial BH3-mimetics therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy modulators sensitizes AML cells to various BH3-mimetics targeting different BCL-2 family members. One such regulator is MFN2, whose protein levels positively correlate with drug resistance in patients with AML. MFN2 overexpression is sufficient to drive resistance to BH3-mimetics in AML. Insensitivity to BH3-mimetics is accompanied by enhanced mitochondria-endoplasmic reticulum interactions and augmented mitophagy flux which acts as a pro-survival mechanism to eliminate mitochondrial damage. Genetic or pharmacologic MFN2 targeting synergizes with BH3-mimetics by impairing mitochondrial clearance and enhancing apoptosis in AML.
PMID: 37088914
ISSN: 2159-8290
CID: 5464912

Juvenile CLN3 disease is a lysosomal cholesterol storage disorder: similarities with Niemann-Pick type C disease

Chen, Jacinda; Soni, Rajesh Kumar; Xu, Yimeng; Simoes, Sabrina; Liang, Feng-Xia; DeFreitas, Laura; Hwang, Robert; Montesinos, Jorge; Lee, Joseph H; Area-Gomez, Estela; Nandakumar, Renu; Vardarajan, Badri; Marquer, Catherine
BACKGROUND:The most common form of neuronal ceroid lipofuscinosis (NCL) is juvenile CLN3 disease (JNCL), a currently incurable neurodegenerative disorder caused by mutations in the CLN3 gene. Based on our previous work and on the premise that CLN3 affects the trafficking of the cation-independent mannose-6 phosphate receptor and its ligand NPC2, we hypothesised that dysfunction of CLN3 leads to the aberrant accumulation of cholesterol in the late endosomes/lysosomes (LE/Lys) of JNCL patients' brains. METHODS:An immunopurification strategy was used to isolate intact LE/Lys from frozen autopsy brain samples. LE/Lys isolated from samples of JNCL patients were compared with age-matched unaffected controls and Niemann-Pick Type C (NPC) disease patients. Indeed, mutations in NPC1 or NPC2 result in the accumulation of cholesterol in LE/Lys of NPC disease samples, thus providing a positive control. The lipid and protein content of LE/Lys was then analysed using lipidomics and proteomics, respectively. FINDINGS/RESULTS:Lipid and protein profiles of LE/Lys isolated from JNCL patients were profoundly altered compared to controls. Importantly, cholesterol accumulated in LE/Lys of JNCL samples to a comparable extent than in NPC samples. Lipid profiles of LE/Lys were similar in JNCL and NPC patients, except for levels of bis(monoacylglycero)phosphate (BMP). Protein profiles detected in LE/Lys of JNCL and NPC patients appeared identical, except for levels of NPC1. INTERPRETATION/CONCLUSIONS:Our results support that JNCL is a lysosomal cholesterol storage disorder. Our findings also support that JNCL and NPC disease share pathogenic pathways leading to aberrant lysosomal accumulation of lipids and proteins, and thus suggest that the treatments available for NPC disease may be beneficial to JNCL patients. This work opens new avenues for further mechanistic studies in model systems of JNCL and possible therapeutic interventions for this disorder. FUNDING/BACKGROUND:San Francisco Foundation.
PMID: 37245481
ISSN: 2352-3964
CID: 5536612