Aggregation of damaged or misfolded proteins is a protective mechanism against proteotoxic stress, abnormalities of which underlie many aging-related diseases. Here, we show that in asymmetrically ...dividing yeast cells, aggregation of cytosolic misfolded proteins does not occur spontaneously but requires new polypeptide synthesis and is restricted to the surface of ER, which harbors the majority of active translation sites. Protein aggregates formed on ER are frequently also associated with or are later captured by mitochondria, greatly constraining aggregate mobility. During mitosis, aggregates are tethered to well-anchored maternal mitochondria, whereas mitochondria acquired by the bud are largely free of aggregates. Disruption of aggregate-mitochondria association resulted in increased mobility and leakage of mother-accumulated aggregates into the bud. Cells with advanced replicative age exhibit gradual decline of aggregates-mitochondria association, likely contributing to their diminished ability to rejuvenate through asymmetric cell division.
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•Aggregation of misfolded cytosolic proteins requires active translation•Aggregation is initiated by newly synthesized polypeptides on the surface of ER•ER-associated aggregation sites are frequently bound or captured by mitochondria•Mitochondria control the dynamics and asymmetric segregation of aggregates
Aggregation of cytosolic misfolded proteins requires new polypeptide synthesis, is restricted to the surface of ER, and is later captured by mitochondria, enabling asymmetric inheritance of protein aggregates during yeast cell division.
Memories are thought to be formed in response to transient experiences, in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA ...binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, we find that the monomeric form of Orb2 represses translation and removes mRNA poly(A) tails, while the oligomeric form enhances translation and elongates the poly(A) tails and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative poly(A) binding protein CG4612 promotes oligomeric Orb2-dependent translation. Our data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory.
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•Drosophila Orb2 has two distinct physical states: monomer and amyloid-like oligomer•The monomeric Orb2 represses, whereas oligomeric Orb2 activates translation•The monomeric Orb2 removes, whereas oligomeric Orb2 protects/elongates poly(A) tail•Two proteins, CG13928 and CG4612, contribute to repression and activation, respectively
The Orb2 protein switches from repressing to activating translation when it forms amyloid-like oligomers, suggesting a possible mechanism by which fleeting experiences create an enduring memory.
Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex ...localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.
During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin ...cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.
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► Heat-induced aggregates undergo random motion, not actin-based retrograde flow ► Hsp104p chaperon clears heat aggregates in the bud through dissolution ► Actin, not formin, contributes to dissolution and motion of heat aggregates ► Aggregates may be retained in aged mothers due to slow diffusion and cell geometry.
In contrast to previous findings, clearance of heat-induced protein aggregates from the bud during yeast cell division occurs primarily through dissolution by the Hsp104p chaperon, and not through retrograde transport toward the mother cell.
Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely ...unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.
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•Mitotic transcription inhibition occurs in early mitosis•P-TEFb is required for mitotic transcriptional activation and release of paused Pol II•Nascent RNA-seq and RNA FISH reveal active transcription at the onset of mitosis•Inhibition of mitotic transcriptional activation delays cell-cycle progression
How transcription is shut down as cells begin to condense chromosomes during mitosis is poorly understood. Liang et al. report the requirement of mitotic transcriptional activation by P-TEFb to release paused Pol II as a prerequisite for this process, and ultimately for proper cell-cycle progression.
Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) ...spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.
Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein ...Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.
The centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present ...in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast, we developed a method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase-coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell-cycle-coupled oscillation of centromeric nucleosome structure in yeast.
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► A new method enables copy measurements of fluorescent proteins in live cells ► Cse4-Cse4 interactions are restricted to anaphase at centromeres ► Cse4 can be deposited at centromeres during anaphase ► Scm3 is absent from Cse4 nucleosomes at anaphase
In vivo single-molecule imaging of yeast cells reveals that centromeric nucleosomes oscillate between tetrameric and octameric forms through the cell cycle.
While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a ...Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.
Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for survival in diverse environments. Organisms inhabiting largely arrhythmic environments, such as caves, ...offer a unique opportunity to study the evolution of circadian rhythms in response to changing ecological pressures. Populations of the Mexican tetra, Astyanax mexicanus, have repeatedly invaded caves from surface rivers, where individuals must contend with perpetual darkness, reduced food availability, and limited fluctuations in daily environmental cues. To investigate the molecular basis for evolved changes in circadian rhythms, we investigated rhythmic transcription across multiple independently-evolved cavefish populations. Our findings reveal that evolution in a cave environment has led to the repeated disruption of the endogenous biological clock, and its entrainment by light. The circadian transcriptome shows widespread reductions and losses of rhythmic transcription and changes to the timing of the activation/repression of core-transcriptional clock. In addition to dysregulation of the core clock, we find that rhythmic transcription of the melatonin regulator aanat2 and melatonin rhythms are disrupted in cavefish under darkness. Mutants of aanat2 and core clock gene rorca disrupt diurnal regulation of sleep in A. mexicanus, phenocopying circadian modulation of sleep and activity phenotypes of cave populations. Together, these findings reveal multiple independent mechanisms for loss of circadian rhythms in cavefish populations and provide a platform for studying how evolved changes in the biological clock can contribute to variation in sleep and circadian behavior.
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