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.
Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full ...structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.
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.
The Arp2/3 complex nucleates the formation of the dendritic actin network at the leading edge of motile cells, but it is still unclear if the Arp2/3 complex plays a critical role in lamellipodia ...protrusion and cell motility. Here, we differentiated motile fibroblast cells from isogenic mouse embryonic stem cells with or without disruption of the ARPC3 gene, which encodes the p21 subunit of the Arp2/3 complex. ARPC3(-/-) fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions, with formin proteins (mDia1 and mDia2) concentrated near their tips. The speed of cell migration, as well as the rates of leading edge protrusion and retraction, were comparable between genotypes; however, ARPC3(-/-) cells exhibited a strong defect in persistent directional migration. This deficiency correlated with a lack of coordination of the protrusive activities at the leading edge of ARPC3(-/-) fibroblasts. These results provide insights into the Arp2/3 complex's critical role in lamellipodia extension and directional fibroblast migration.
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.
Actin filaments and myosin II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Here we show that in budding yeast, actin filament depolymerization ...plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuate actomyosin ring constriction. Deletion of myosin II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin II motor activity. Model simulations based on experimental measurements support the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time regardless of the initial ring size, as originally reported for C. elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to different ploidies.
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► Actin filament depolymerization plays a major role in budding yeast cytokinesis ► Genetic or chemical stabilization of actin filaments impairs ring constriction ► Quantitative model of contractile stress generation via actin depolymerization ► Model predicts invariant contraction time regardless of the initial ring size
The total contraction time of cytokinesis appears invariant, but how is actomyosin contractility scaled according to cell size? Pinto et al. find that, during budding yeast cytokinesis, myosin II generates force primarily via actin depolymerization rather than contractility, and they discuss the implications of this for scalability.
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.
Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales ...relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein’s self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.
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•Distributed amphifluoric FRET (DAmFRET) quantifies nucleation in living cells•DAmFRET rapidly distinguishes prion-like from non-prion phase transitions•Sequence-intrinsic features determine concentration dependence of nucleation barriers•Prion cross-seeding occurs by conformational templating, not by condensation
Prion phenomena result from protein phase separations that are rate limited by nucleation. Here, Khan et al. introduce a method to quantify nucleation in living cells. By comparing diverse proteins with and without prion behavior, they show that the kinetic barrier to nucleation derives from structural order in the new phase.
The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating ...multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.
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