Assessing Autophagy in Muscle Stem Cells.

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity's relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.Work in the authors’ laboratory has been supported by MINECO-Spain (RTI2018-096068), ERC-2016-AdG- 741966, LaCaixa-HEALTH-HR17-00040, MDA, Fundació LaMarató/TV3, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain, as well as María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370), Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). SC was supported by a Predoctoral Fellowship from Ayudas para la formación y contratación de personal investigador novel (FI) – AGAUR (Barcelona, Spain), IR-P was supported by a Predoctoral Fellowship from Programa de Formación de Personal Investigador (Spain), and XH was supported by a Severo-Ochoa Pre-doctoral Fellowship (Spain).S

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe Smoothelin-like 2 (SMTNL2) function, a member of the smooth-muscle related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during the development of multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of Coronin-1B. While Coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to Coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular corte

FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.The authors acknowledge funding from MINECO-Spain (grant no. RTI2018-096068), ERC2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain to P.M.-C; María-de-Maeztu-Program for Units of Excellence to UPF (grant no. MDM-2014-0370) and the Severo-Ochoa-Program for Centers of Excellence to CNIC (grant no. SEV-2015-0505). This work was also supported by NIAMS IRP through NIH grants nos AR041126 and AR041164 to V.S. and utilized computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov); ASI, Ricerca Finalizzata, Ateneo Sapienza to A.M.; AIRC (grant no. 23257); ASI (grant no. MARS-PRE, DC-VUM-2017-006); H2020-MSCA-RISE-2014 (645648) to M.S. and a FNR core grant (grant no. C15/BM/10397420) to A.d.S. L.G.P. was partially supported by an FPI fellowship and an EMBO fellowship (grant no. ALTF 420-2017); and S.C., X.H. and V.M. by FI, Severo-Ochoa and PFI Fellowships (Spain), respectively

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.Work in the PMC laboratory was supported by Spanish Ministerio de Ciencia e Innovación ( RTI2018-096068 to P.M.-C. and E.P), ERC - 2016-AdG-741966 , LaCaixa - HEALTH-HR17-00040 , MDA , UPGRADE-H2020-825825 , AFM-Telethon , DPP-Spain , Fundació La Marató TV3-80/19-202021 to P.M.-C; Fundació La Marató TV3-137/38-202033 to A.L.S.; partly supported by Milky Way Research Foundation (MWRF) to P.M.-C; Severo Ochoa Program for Centers of Excellence to CNIC ( SEV-2015-0505 ) and Maria de Maeztu Program for Units of Excellence to UPF ( MDM-2014-0370 ). Work in the JAE laboratory was supported by Ministerio de Ciencia e Innovacion ( RTI2018-099357-B-I00 , RED2018-102576-T ), Human Frontier Science Program HFSP ( RGP0016/2018 ), Centro de Investigación Biomédica en Red en Fragilidad y Envejecimento Saludable ( CIBERFES16/10/00282 ), and Leduq Foundation award ( REDOX-17CVD04 ). Work in JMV laboratory was supported by the Spanish Ministerio de Ciencia e Innovación ( RTI2018-100695-B-I00 ), Spanish Junta de Andalucía ( P18-RT-4264 , 1263735-R and BIO-276 ), the FEDER Funding Program from the European Union , and Universidad de Córdoba . The authors are indebted to the personnel from the Servicio Centralizado de Apoyo a la Investigación (SCAI; University of Córdoba) for technical support with the transmission electron microscope. Work in MS laboratory was funded by the Italian Assoc. for Cancer Research ( AIRC IG-D17388 and ID23257 ) and ASI (MARS-PRE, project DC-VUM-2017-006). X.H., S.C., I.R.-P, and A.C were supported by Severo Ochoa PFI , PI , FPI , and H2020 Marie Skłodowska-Curie Actions predoctoral fellowships, respectively. P.H.-A was supported by Juan de la Cierva-Incorporación fellowship

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.This work was supported by General Research Funds (GRF) from the Research Grants Council (RGC) of the Hong Kong Special Administrative Region (14115319, 14100018, 14100620, 14106117 and 14106521 to H.W.; 14120420, 14116918, and 14120619 to H.S.); Guangdong Natural Science Foundation from Guangdong Basic and Applied Basic Research Foundation to X.C. (Project code: 2019A1515010670); the National Natural Science Foundation of China (NSFC) to H.W. (Project code: 31871304); Collaborative Research Fund (CRF) from RGC to H.W. (C6018-19GF); CUHK Direct Grant for Research to H.W. (Project code: 4054482); NSFC/RGC Joint Research Scheme to H.S. (Project code: N_CUHK 413/18); Hong Kong Epigenomics Project (EpiHK) Fund to H.W. and H.S.; Area of Excellence Scheme (AoE) from RGC (Project number: AoE/M-402/20). Work in PMC laboratory was supported by Spanish Ministry of Science, Innovation and Universities, Spain (grants RTI2018-096068-B-I00 and SAF 2015-70270-REDT, a María de Maeztu Unit of Excellence award to UPF [MDM-2014-0370], and a Severo Ochoa Center of Excellence award to the CNIC [SEV-2015-0505]), ERC-2016-AdG-741966, La Caixa-HEALTH (HR17-00040), MDA, UPGRADE-H2020-825825, AFM and DPP-E. S.C. is recipient of a FI fellowship from AGAUR. Work in CKK laboratory was supported by Shenzhen Basic Research Project (JCYJ20180507181642811), Research Grants Council of the Hong Kong SAR (CityU 11101519, CityU 11100218, N_CityU110/17, CityU 21302317), Croucher Foundation Project (9509003, 9500030), State Key Laboratory of Marine Pollution Director Discretionary Fund, City University of Hong Kong projects (6000711, 7005503, 9680261, and 9667222 to C.K.K.). Work in YX laboratory was supported by the National Natural Science Foundation of China (32025008 and 91940306 to Y.X.)

Higgs boson potential at colliders: status and perspectives

This document summarises the current theoretical and experimental status of the di-Higgs boson production searches, and of the direct and indirect constraints on the Higgs boson self-coupling, with the wish to serve as a useful guide for the next years. The document discusses the theoretical status, including state-of-the-art predictions for di-Higgs cross sections, developments on the effective field theory approach, and studies on specific new physics scenarios that can show up in the di-Higgs final state. The status of di-Higgs searches and the direct and indirect constraints on the Higgs self-coupling at the LHC are presented, with an overview of the relevant experimental techniques, and covering all the variety of relevant signatures. Finally, the capabilities of future colliders in determining the Higgs self-coupling are addressed, comparing the projected precision that can be obtained in such facilities. The work has started as the proceedings of the Di-Higgs workshop at Colliders, held at Fermilab from the 4th to the 9th of September 2018, but it went beyond the topics discussed at that workshop and included further developments. Part III of the document reviews the capabilities of future colliders to establish the the size of Higgs self-coupling both qualitatively and quantitatively