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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

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

Hedgehog and PDGF Signaling Intersect During Postnatal Lung Development

Yie, Ting-An; Loomis, Cynthia A; Nowatzky, Johannes; Khodadadi-Jamayran, Alireza; Lin, Ziyan; Cammer, Michael; Barnett, Clea; Mezzano, Valeria; Alu, Mark; Novick, Jackson A; Munger, John S; Kugler, Matthias C
Normal lung development critically depends on Hedgehog (HH) and Platelet-derived growth factor (PDGF) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH and PDGF signaling and their impact/convergence on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of Pdgfa and Pdgfra knockouts during secondary alveolar septation. Utilizing a dual signaling reporter, Gli1IZ;PdgfraEGFP
PMID: 36693140
ISSN: 1535-4989
CID: 5419542

Dedifferentiation maintains melanocyte stem cells in a dynamic niche

Sun, Qi; Lee, Wendy; Hu, Hai; Ogawa, Tatsuya; De Leon, Sophie; Katehis, Ioanna; Lim, Chae Ho; Takeo, Makoto; Cammer, Michael; Taketo, M Mark; Gay, Denise L; Millar, Sarah E; Ito, Mayumi
For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.
PMID: 37076619
ISSN: 1476-4687
CID: 5464522

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death

Matsuzawa-Ishimoto, Yu; Yao, Xiaomin; Koide, Akiko; Ueberheide, Beatrix M; Axelrad, Jordan E; Reis, Bernardo S; Parsa, Roham; Neil, Jessica A; Devlin, Joseph C; Rudensky, Eugene; Dewan, M Zahidunnabi; Cammer, Michael; Blumberg, Richard S; Ding, Yi; Ruggles, Kelly V; Mucida, Daniel; Koide, Shohei; Cadwell, Ken
Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.
PMID: 36198790
ISSN: 1476-4687
CID: 5351622

Interleukin-17 governs hypoxic adaptation of injured epithelium

Konieczny, Piotr; Xing, Yue; Sidhu, Ikjot; Subudhi, Ipsita; Mansfield, Kody P; Hsieh, Brandon; Biancur, Douglas E; Larsen, Samantha B; Cammer, Michael; Li, Dongqing; Landén, Ning Xu; Loomis, Cynthia; Heguy, Adriana; Tikhonova, Anastasia N; Tsirigos, Aristotelis; Naik, Shruti
Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt+ (RORγt+) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.
PMID: 35709248
ISSN: 1095-9203
CID: 5268732

Metabolic reprogramming of tumor-associated macrophages by collagen turnover promotes fibrosis in pancreatic cancer

LaRue, Madeleine M; Parker, Seth; Puccini, Joseph; Cammer, Michael; Kimmelman, Alec C; Bar-Sagi, Dafna
SignificanceThe highly desmoplastic and immunosuppressive microenvironment of pancreatic tumors is a major determinant of the aggressive nature and therapeutic resistance of pancreatic cancer. Therefore, improving our understanding of the mechanisms that regulate the composition and function of the pancreatic tumor microenvironment is critical for the design of intervention strategies for this devastating malignancy. This study identifies a modality for the reprogramming of tumor-associated macrophages involving collagen scavenging followed by a metabolic switch toward a profibrotic paracrine phenotype. These findings establish a molecular framework for the elucidation of regulatory processes that could be harnessed to mitigate the stroma-dependent protumorigenic effects in pancreatic cancer.
PMID: 35412885
ISSN: 1091-6490
CID: 5201902