RESUMO
Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene-expression patterns have been reported: the "environmental stress response" (ESR) and the "common aneuploidy gene-expression" (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate this controversy. We show that the CAGE signature is not an aneuploidy-specific gene-expression signature but the result of normalizing the gene-expression profile of actively proliferating aneuploid cells to that of euploid cells grown into stationary phase. Because growth into stationary phase is among the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene-expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells exhibit the ESR. We further show that the ESR causes selective ribosome loss in aneuploid cells, providing an explanation for the decreased cellular density of aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than the CAGE signature, in response to aneuploidy-induced cellular stresses, resulting in selective ribosome loss. We propose that the ESR serves two purposes in aneuploid cells: protecting cells from aneuploidy-induced cellular stresses and preventing excessive cellular enlargement during slowed cell cycles by down-regulating translation capacity.
Assuntos
Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Aneuploidia , Meio Ambiente , Regulação Fúngica da Expressão Gênica , RNA Fúngico/genética , RNA Fúngico/metabolismo , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved.
RESUMO
Aneuploid yeast cells are in a chronic state of proteotoxicity, yet do not constitutively induce the cytosolic unfolded protein response, or heat shock response (HSR) by heat shock factor 1 (Hsf1). Here, we demonstrate that an active environmental stress response (ESR), a hallmark of aneuploidy across different models, suppresses Hsf1 induction in models of single-chromosome gain. Furthermore, engineered activation of the ESR in the absence of stress was sufficient to suppress Hsf1 activation in euploid cells by subsequent heat shock while increasing thermotolerance and blocking formation of heat-induced protein aggregates. Suppression of the ESR in aneuploid cells resulted in longer cell doubling times and decreased viability in the presence of additional proteotoxicity. Last, we show that in euploids, Hsf1 induction by heat shock is curbed by the ESR. Strikingly, we found a similar relationship between the ESR and the HSR using an inducible model of aneuploidy. Our work explains a long-standing paradox in the field and provides new insights into conserved mechanisms of proteostasis with potential relevance to cancers associated with aneuploidy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Adaptação Biológica/genética , Aneuploidia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas/genéticaRESUMO
BACKGROUND: Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections. NEW METHOD: We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy. RESULTS: Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable. COMPARISON WITH EXISTING METHOD: RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections. CONCLUSIONS: RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.