RESUMEN
Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.
Asunto(s)
Actinas , Septinas , Humanos , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Microscopía , Septinas/análisisRESUMEN
Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Septinas , Fibras de Estrés , Humanos , Proteínas Asociadas a Microtúbulos , Microtúbulos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismo , Fibras de Estrés/metabolismoRESUMEN
iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis , Proteínas Represoras/metabolismo , Huso Acromático/metabolismo , Secuencias de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica , Proteínas Represoras/químicaRESUMEN
Living species are chronically exposed to environmental ionizing radiations from sources that can be overexpressed by nuclear accidents. In invertebrates, reproduction is the most radiosensitive studied endpoint, likely to be connected with aging. Surprisingly, aging is a sparsely investigated endpoint after chronic ionizing radiation, whereas understanding it is of fundamental interest in biology and medicine. Indeed, aging and aging-related diseases (e.g., cancer and degenerative diseases) cause about 90% of deaths in developed countries. Therefore, glp-1 sterile Caenorhabditis elegans nematode was used to assess the impact of chronic gamma irradiation on the lifespan. Analyses were performed, at the individual level, on aging and, in order to delve deeper into the mechanisms, at the molecular level, on oxidative damage (carbonylation), biomolecules (lipids, proteins and nucleic acids) and their colocalization. We observed that ionizing radiation accelerates aging (whatever the duration (3-19 days)/dose (0.5-24â¯Gy)/dose rate (7 and 52â¯mGyâ¯h-1) tested) leading to a longevity value equivalent to that of wt nematode (â¼25-30 days). Moreover, the level of protein oxidative damage (carbonylation) turned out to be good cellular biomarker of aging, since it increases with age. Conversely, chronic radiation treatments reduced carbonylation levels and induced neutral lipid catabolism whatever the dose rate and the final delivered dose. Finally, under some conditions a lipid-protein colocalization without any carbonyl was observed; this could be linked to yolk accumulation in glp-1 nematodes. To conclude, we noticed through this study a link between chronic gamma exposure, lifespan shortening and lipid level decrease associated with a decrease in the overall carbonylation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Rayos gamma/efectos adversos , Longevidad/efectos de la radiación , Mutación , Receptores Notch/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Tolerancia a RadiaciónRESUMEN
Together with nematodes and rotifers, tardigrade belong to micrometazoans that can cope with environmental extremes such as UV and solar radiations, dehydration, supercooling or overheating. Tardigrade can resist the harshest conditions by turning to cryptobiosis, an anhydrobiotic state that results from almost complete dehydration and is characterized by an ametabolic status. Although reports have challenged the molecular basis of the mechanisms underlying genomic injury resistance, little is yet known regarding the possible involvement of other tardigrade macromolecules in injury during a stress experience. In this report, we show that the tardigrade Hypsibius exemplaris can accumulate molecular damages by means of in situ detection of carbonyls. Furthermore, we demonstrate that living tardigrade can accumulate carbonylation. Finally, we reveal that anhydrobiotic tardigrade can be constitutively affected by carbonylation that marks aging in other metazoans.
Asunto(s)
Aldehídos/metabolismo , Cetonas/metabolismo , Tardigrada/química , Aldehídos/química , Animales , Cetonas/química , Microscopía Confocal , Tardigrada/crecimiento & desarrollo , Tardigrada/efectos de la radiación , Factores de Tiempo , Rayos UltravioletaRESUMEN
Species are chronically exposed to ionizing radiation, a natural phenomenon which can be enhanced by human activities. The induced toxicity mechanisms still remain unclear and seem depending on the mode of exposure, i.e. acute and chronic. To better understand these phenomena, studies need to be conducted both at the subcellular and individual levels. Proteins, functional molecules in organisms, are the targets of oxidative damage (especially via their carbonylation (PC)) and are likely to be relevant biomarkers. After exposure of Caenorhabditis elegans to either chronic or acute γ rays we showed that hatching success is impacted after acute but not after chronic irradiation. At the molecular level, the carbonylated protein level in relation with dose was slightly different between acute and chronic exposure whereas the proteolytic activity is drastically modified. Indeed, whereas the 20S proteasome activity is inhibited by acute irradiation from 0.5 Gy, it is activated after chronic irradiation from 1 Gy. As expected, the 20S proteasome activity is mainly modified by irradiation whereas the 26S and 30S activity are less changed. This study provides preliminaries clues to understand the role of protein oxidation and proteolytic activity in the radiation-induced molecular mechanisms after chronic versus acute irradiation in C. elegans.
Asunto(s)
Caenorhabditis elegans/efectos de la radiación , Rayos gamma , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Relación Dosis-Respuesta en la Radiación , Cinética , Carbonilación Proteica/efectos de la radiación , Proteolisis/efectos de la radiación , Reproducción/efectos de la radiaciónRESUMEN
All key biological macromolecules are susceptible to carbonylation - an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.