Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

• Klíčová slova
• C. elegans, MAP9, axoneme, cilia, dynein, kinesin, microtubule, microtubule doublet, microtubule-associated protein, polyglutamylation,
• MeSH
• axonema * metabolismus   ultrastruktura   MeSH
• Caenorhabditis elegans * metabolismus   MeSH
• cilie metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• pohyb MeSH
• savci MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Map9 protein, mouse MeSH Prohlížeč
• tubulin MeSH

Under adverse conditions such as shade or elevated temperatures, cotyledon expansion is reduced and hypocotyl growth is promoted to optimize plant architecture. The mechanisms underlying the repression of cotyledon cell expansion remain unknown. Here, we report that the nuclear abundance of the BES1 transcription factor decreased in the cotyledons and increased in the hypocotyl in Arabidopsis thaliana under shade or warmth. Brassinosteroid levels did not follow the same trend. PIF4 and COP1 increased their nuclear abundance in both organs under shade or warmth. PIF4 directly bound the BES1 promoter to enhance its activity but indirectly reduced BES1 expression. COP1 physically interacted with the BES1 protein, promoting its proteasome degradation in the cotyledons. COP1 had the opposite effect in the hypocotyl, demonstrating organ-specific regulatory networks. Our work indicates that shade or warmth reduces BES1 activity by transcriptional and post-translational regulation to inhibit cotyledon cell expansion.

• Klíčová slova
• COP1, PIF4, brassinosteroids, growth (plants), plant hormones, shade avoidance, thermomorphogenesis,
• MeSH
• Arabidopsis * metabolismus   MeSH
• brassinosteroidy metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• hypokotyl metabolismus   MeSH
• proteiny huseníčku * metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BES1 protein, Arabidopsis MeSH Prohlížeč
• brassinosteroidy MeSH
• DNA vazebné proteiny MeSH
• proteiny huseníčku * MeSH

When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes.

• Klíčová slova
• T cells, Wiskott-Aldrich syndrome protein, actin cytoskeleton, ameboid motility, cell migration, confinement, dendritic cells, leukocytes, mechanical load, mechanosensing,
• MeSH
• aktiny metabolismus   fyziologie   MeSH
• biomechanika fyziologie   MeSH
• buněčné linie MeSH
• cytoskeletální proteiny metabolismus   MeSH
• komplex proteinů 2-3 souvisejících s aktinem metabolismus   fyziologie   MeSH
• leukocyty fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pohyb buněk fyziologie   MeSH
• protein 3 související s aktinem metabolismus   MeSH
• protein Wiskottova-Aldrichova syndromu genetika   metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• cytoskeletální proteiny MeSH
• komplex proteinů 2-3 souvisejících s aktinem MeSH
• protein 3 související s aktinem MeSH
• protein Wiskottova-Aldrichova syndromu MeSH

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

• Klíčová slova
• DeltaSILAC, data-independent acquisition, mass spectrometry, phosphomodiform, phosphorylation, protein lifetime, protein turnover, proteomics, pulse SILAC,
• MeSH
• buněčný cyklus fyziologie   MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• glutamáty metabolismus   MeSH
• hmotnostní spektrometrie metody   MeSH
• izotopové značení metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• peptidy metabolismus   MeSH
• peroxiredoxin VI chemie   metabolismus   MeSH
• proteolýza * MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• sestřihové faktory chemie   metabolismus   MeSH
• signální transdukce genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cyklin-dependentní kinasy MeSH
• fosfoproteiny MeSH
• glutamáty MeSH
• peptidy MeSH
• peroxiredoxin VI MeSH
• PRDX6 protein, human MeSH Prohlížeč
• proteom MeSH
• sestřihové faktory MeSH
• SF3B1 protein, human MeSH Prohlížeč

Cell polarity is a key feature in the development of multicellular organisms. For instance, asymmetrically localized plasma-membrane-integral PIN-FORMED (PIN) proteins direct transcellular fluxes of the phytohormone auxin that govern plant development. Fine-tuned auxin flux is important for root protophloem sieve element differentiation and requires the interacting plasma-membrane-associated BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) proteins. We observed "donut-like" polar PIN localization in developing sieve elements that depends on complementary, "muffin-like" polar localization of BRX and PAX. Plasma membrane association and polarity of PAX, and indirectly BRX, largely depends on phosphatidylinositol-4,5-bisphosphate. Consistently, mutants in phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) display protophloem differentiation defects similar to brx mutants. The same PIP5Ks are in complex with BRX and display "muffin-like" polar localization. Our data suggest that the BRX-PAX module recruits PIP5Ks to reinforce PAX polarity and thereby the polarity of all three proteins, which is required to maintain a local PIN minimum.

• Klíčová slova
• Arabidopsis, DRP1A, PIP5K1, PIP5K2, endocytosis, phloem, polar auxin transport, polarity, protophloem, root,
• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• buněčná diferenciace * MeSH
• buněčná membrána metabolismus   MeSH
• fosfatasy genetika   metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• mutace MeSH
• polarita buněk * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-phosphatidylinositol-4-phosphate 5-kinase MeSH Prohlížeč
• BREVIS RADIX protein, Arabidopsis MeSH Prohlížeč
• CVP2 protein, Arabidopsis MeSH Prohlížeč
• fosfatasy MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• proteiny huseníčku MeSH
• transkripční faktory paired box MeSH

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

• Klíčová slova
• AAK1, AP2 endocytic adaptor, NECAP, NMR, Numb-associated kinases (NAK), SNX9, TIRF, clathrin-mediated endocytosis, crystallography, regulation by phosphorylation,
• MeSH
• adaptorový proteinový komplex - alfa-podjednotky genetika   MeSH
• adaptorový proteinový komplex 2 genetika   metabolismus   MeSH
• endocytóza genetika   MeSH
• fosforylace genetika   MeSH
• klathrin genetika   metabolismus   MeSH
• klathrinové vezikuly genetika   metabolismus   MeSH
• lidé MeSH
• potažené jamky v buněčné membráně genetika   metabolismus   MeSH
• třídící nexiny genetika   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorový proteinový komplex - alfa-podjednotky MeSH
• adaptorový proteinový komplex 2 MeSH
• klathrin MeSH
• NECAP1 protein, human MeSH Prohlížeč
• SNX9 protein, human MeSH Prohlížeč
• třídící nexiny MeSH

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.

• Klíčová slova
• Arabidopsis, biosensor, endocytosis, lipid signaling, membrane biology, phosphatidic acid, phosphatidylserine, phosphoinositides, plant cell biology, vesicular trafficking,
• MeSH
• Arabidopsis růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfatidylseriny metabolismus   MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• kyseliny fosfatidové metabolismus   MeSH
• organely MeSH
• proteiny huseníčku metabolismus   MeSH
• signální transdukce MeSH
• statická elektřina * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylinositolfosfáty MeSH
• fosfatidylseriny MeSH
• kyseliny fosfatidové MeSH
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• proteiny huseníčku MeSH

Binding of the transcriptional co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines postnatally. While investigating factors that limit YAP's postnatal mitogenic activity, we found that the CM-enriched TEAD1 binding protein VGLL4 inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulated its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer.

• Klíčová slova
• Hippo-YAP pathway, TEAD1, VGLL4, acetylation, cardiac, cardiomyocyte, degradation, necrosis, proliferation,
• MeSH
• acetylace MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• lidé MeSH
• novorozená zvířata MeSH
• potkani Wistar MeSH
• proliferace buněk MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinové domény MeSH
• proteiny buněčného cyklu MeSH
• sekvence aminokyselin MeSH
• signální dráha Hippo MeSH
• signální proteiny YAP MeSH
• signální transdukce * MeSH
• srdce růst a vývoj   MeSH
• srdeční selhání metabolismus   patologie   MeSH
• stabilita proteinů MeSH
• stárnutí metabolismus   MeSH
• transkripční faktory TEA domény MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• DNA vazebné proteiny MeSH
• fosfoproteiny MeSH
• protein-serin-threoninkinasy MeSH
• proteiny buněčného cyklu MeSH
• signální proteiny YAP MeSH
• Tead1 protein, mouse MeSH Prohlížeč
• transkripční faktory TEA domény MeSH
• transkripční faktory MeSH
• VGLL4 protein, mouse MeSH Prohlížeč
• Yap1 protein, mouse MeSH Prohlížeč

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.

• MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• fibroblastové růstové faktory metabolismus   MeSH
• mezoderm cytologie   metabolismus   MeSH
• moláry cytologie   embryologie   metabolismus   MeSH
• morfogeneze fyziologie   MeSH
• myši MeSH
• odontogeneze fyziologie   MeSH
• pohyb buněk fyziologie   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zuby cytologie   embryologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fibroblastové růstové faktory MeSH

Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.

• MeSH
• Arabidopsis cytologie   fyziologie   MeSH
• cytokineze fyziologie   MeSH
• cytoplazmatické vezikuly metabolismus   MeSH
• exocytóza fyziologie   MeSH
• mikrotubuly metabolismus   MeSH
• molekulární modely MeSH
• proteiny huseníčku metabolismus   MeSH
• růstová ploténka cytologie   metabolismus   MeSH
• vezikulární transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• proteiny huseníčku MeSH
• transport protein particle, TRAPP MeSH Prohlížeč
• vezikulární transportní proteiny MeSH