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Myeloid‐resident neuropilin‐1 promotes choroidal neovascularization while mitigating inflammation

2021-04-19T00:00

Age‐related macular degeneration (AMD) in its various forms is a leading cause of blindness in industrialized countries. Here, we provide evidence that ligands for neuropilin‐1 (NRP1), such as Semaphorin 3A and VEGF‐A, are elevated in the vitreous of patients with AMD at times of active choroidal neovascularization (CNV). We further demonstrate that NRP1‐expressing myeloid cells promote and maintain CNV. Expression of NRP1 on cells of myeloid lineage is critical for mitigating production of inflammatory factors such as IL6 and IL1β. Therapeutically trapping ligands of NRP1 with an NRP1‐derived trap reduces CNV. Collectively, our findings identify a role for NRP1‐expressing myeloid cells in promoting pathological angiogenesis during CNV and introduce a therapeutic approach to counter neovascular AMD.

A population of innate immune myeloid cells expressing NRP1 receptor invades the retina where it drives and maintains pathological neovascularization during age‐related macular degeneration (AMD). A recombinant NRP1‐derived trap prevents choroidal neovascularization‐associated pathological angiogenesis.

Myeloid‐resident neuropilin‐1 promotes choroidal neovascularization while mitigating inflammation

2021-04-19T00:00

Centromeric transcription maintains centromeric cohesion in human cells

2021-04-21T00:00

Centromeric transcription is critical for proper centromere function, but its exact role remains elusive. By manipulating the activity of centromeric transcription, Chen et al. report that a major function of centromeric transcription in human cells is to maintain centromeric cohesion.

Centromeric transcription has been shown to play an important role in centromere functions. However, lack of approaches to specifically manipulate centromeric transcription calls into question that the proposed functions are a direct consequence of centromeric transcription. By monitoring nascent RNAs, we found that several transcriptional inhibitors exhibited distinct, even opposing, efficacies on the suppression of ongoing gene and centromeric transcription in human cells, whereas under the same conditions, total centromeric RNAs were changed to a lesser extent. The inhibitor suppressing ongoing centromeric transcription weakened centromeric cohesion, whereas the inhibitor increasing ongoing centromeric transcription strengthened centromeric cohesion. Furthermore, expression of CENP-B DNA-binding domain or CENP-B knockdown moderately increased centromeric transcription without altering gene transcription; as a result, centromeric cohesion was accordingly strengthened. Targeting of the Kox1-KRAB domain with CENP-B DB to centromeres specifically decreased centromeric transcription and weakened centromeric cohesion. Thus, based on these findings, we propose that a major function of centromeric transcription is to maintain centromeric cohesion in human cells.

Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioral outcomes

Sensory cues in the natural environment predict reward or punishment, important for survival. For example, the ability to detect attractive tastes indicating palatable food is essential for foraging while the recognition of inedible substrates prevents harm. While some of these sensory responses are innate, they can undergo fundamental changes due to prior experience associated with the stimulus. However, the mechanisms underlying such behavioral switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis1, 2, 3 to address the question of how an anticipated aversive outcome reverses the behavioral response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal⁴^,⁵), which is inhibited by sucrose to allow a feeding response. After multi-trial aversive associative conditioning, pairing sucrose with strong tactile stimuli to the head, PlB’s firing rate increases in response to sucrose application to the lips and the feeding response is suppressed; this learned response is reversed by the photoinactivation of a single PlB. A learning-induced persistent change in the cellular properties of PlB that results in an increase rather than a decrease in its firing rate in response to sucrose provides a neurophysiological mechanism for this behavioral switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network⁵^,⁶ does not mediate the conditioned suppression of feeding, but its facilitated output contributes to the sensitization of the withdrawal response.

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Anticipation of an aversive outcome reverses the behavioral response to food.

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The switching mechanism relies on an interneuron extrinsic to the feeding network.

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Aversive learning causes persistent physiological change in this interneuron.

Pirger et al. demonstrate that persistent modulation of an inhibitory interneuron extrinsic to the feeding system can switch the behavioral response so that a previously salient food stimulus elicits an aversive response. Anticipation of punishment associated with food induces excitation of an interneuron that inhibits the feeding network.

MICOS subcomplexes assemble independently on the mitochondrial inner membrane in proximity to ER contact sites

2020-10-07T00:00

Tirrell et al. determine that subcomplexes of the cristae-organizing MICOS complex assemble independently of one another on the mitochondrial inner membrane in proximity to contact sites between mitochondria and the endoplasmic reticulum.

MICOS is a conserved multisubunit complex that localizes to mitochondrial cristae junctions and organizes cristae positioning within the organelle. MICOS is organized into two independent subcomplexes; however, the mechanisms that dictate the assembly and spatial positioning of each MICOS subcomplex are poorly understood. Here, we determine that MICOS subcomplexes target independently of one another to sites on the inner mitochondrial membrane that are in proximity to contact sites between mitochondria and the ER. One subcomplex, composed of Mic27/Mic26/Mic10/Mic12, requires ERMES complex function for its assembly. In contrast, the principal MICOS component, Mic60, self-assembles and localizes in close proximity to the ER through an independent mechanism. We also find that Mic60 can uniquely redistribute adjacent to forced mitochondria–vacuole contact sites. Our data suggest that nonoverlapping properties of interorganelle contact sites provide spatial cues that enable MICOS assembly and ultimately lead to proper physical and functional organization of mitochondria.

Conserved actin machinery drives microtubule-independent motility and phagocytosis in Naegleria

2020-09-22T00:00

Naegleria diverged from the “yeast-to-human” lineage >1 billion years ago. Velle and Fritz-Laylin found that Naegleria, which lacks interphase microtubules, has a robust actin cytoskeletal repertoire and uses conserved, Arp2/3–derived actin networks to drive cell crawling and enhance phagocytosis. These findings support an evolutionarily ancient origin for these phenotypes.

Much of our understanding of actin-driven phenotypes in eukaryotes has come from the “yeast-to-human” opisthokont lineage and the related amoebozoa. Outside of these groups lies the genus Naegleria, which shared a common ancestor with humans >1 billion years ago and includes the “brain-eating amoeba.” Unlike nearly all other known eukaryotic cells, Naegleria amoebae lack interphase microtubules; this suggests that actin alone drives phenotypes like cell crawling and phagocytosis. Naegleria therefore represents a powerful system to probe actin-driven functions in the absence of microtubules, yet surprisingly little is known about its actin cytoskeleton. Using genomic analysis, microscopy, and molecular perturbations, we show that Naegleria encodes conserved actin nucleators and builds Arp2/3–dependent lamellar protrusions. These protrusions correlate with the capacity to migrate and eat bacteria. Because human cells also use Arp2/3–dependent lamellar protrusions for motility and phagocytosis, this work supports an evolutionarily ancient origin for these processes and establishes Naegleria as a natural model system for studying microtubule-independent cytoskeletal phenotypes.

Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy

2021-03-11T00:00

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1, 2, 3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4, 5, 6, 7 Different cell types position their nuclei away from the leading edge during cell migration.8, 9, 10, 11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12, 13, 14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15, 16, 17, 18, 19, 20, 21, 22 and the actin regulator Eps8L223, 24, 25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

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Ctdnep1 and Eps8L2 are required for nuclear positioning and TAN lines formation

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Ctdnep1 directly interacts with Eps8L2 for nuclear movement and cell migration

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Ctdnep1-Eps8L2 interaction regulates dorsal actin organization

Nucleo-cytoskeletal connections are crucial for nuclear positioning. Calero-Cuenca et al. show that Ctdnep1 and Eps8L2 interact to regulate perinuclear actin cables organization promoting transmembrane actin-associated nuclear (TAN) lines formation. Ctdnep1-Eps8L2 interaction is required for nuclear positioning and proper cell migration.

Malaria parasites differentially sense environmental elasticity during transmission

2021-03-05T00:00

Transmission of malaria‐causing parasites to and by the mosquito relies on active parasite migration and constitutes bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 h on soft substrates. In contrast, sporozoites evolved more short‐lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion to soft endothelial cells on their long way to the liver. Hence, the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter.

Motility is essential for malaria parasites to infect their hosts. Here, new assays show that Plasmodium adapted to the different elastic properties of the physical environments it encounters. Most strikingly, Plasmodium sporozoites avoid migration on vascular cells to reach the liver.

femaleless Controls Sex Determination and Dosage Compensation Pathways in Females of Anopheles Mosquitoes

The insect sex determination and the intimately linked dosage compensation pathways represent a challenging evolutionary puzzle that has been solved only in Drosophila melanogaster. Analyses of orthologs of the Drosophila genes identified in non-drosophilid taxa¹^,² revealed that evolution of sex determination pathways is consistent with a bottom-up mode,³ where only the terminal genes within the pathway are well conserved. doublesex (dsx), occupying a bottom-most position and encoding sex-specific proteins orchestrating downstream sexual differentiation processes, is an ancient sex-determining gene present in all studied species.²^,⁴^,⁵ With the exception of lepidopterans, its female-specific splicing is known to be regulated by transformer (tra) and its co-factor transformer-2 (tra2).6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Here we show that in the African malaria mosquito Anopheles gambiae, a gene, which likely arose in the Anopheles lineage and which we call femaleless (fle), controls sex determination in females by regulating splicing of dsx and fruitless (fru; another terminal gene within a branch of the sex determination pathway). Moreover, fle represents a novel molecular link between the sex determination and dosage compensation pathways. It is necessary to suppress activation of dosage compensation in females, as demonstrated by the significant upregulation of the female X chromosome genes and a correlated female-specific lethality, but no negative effect on males, in response to fle knockdown. This unexpected property, combined with a high level of conservation in sequence and function in anopheline mosquitoes, makes fle an excellent target for genetic control of all major vectors of human malaria.

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fle is a new sex determination pathway element conserved in Anopheles mosquitoes

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fle may have originated in the Anopheles lineage and is highly conserved in Anopheles

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fle suppresses activation of dosage compensation in females

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Depletion of fle transcripts is lethal or otherwise deleterious to females

Krzywinska et al. identify a new element of the sex determination pathway in Anopheles. femaleless (fle), in addition to controlling splicing of dsx and fru, is essential for suppression of dosage compensation and viability of females. Female-deleterious effects upon fle knockdown make fle a promising target for control of malaria mosquitoes.

Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator

2020-07-16T00:00

Gómez Acuña et al. show that human Argonaute-1 (AGO1) binds genome wide to estrogen receptor α sites at chromatin and acts as a coactivator of estrogen-mediated transcriptional activation. This new role for AGO1 does not depend on binding to small RNAs.

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA–mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.

The function of GORASPs in Golgi apparatus organization in vivo

2020-06-23T00:00

Grond et al. show that GRASPs or GORASPs do not have a role in stacking of Golgi cisternae in vivo but instead are required for linking cisternal rims to likely control flux of transport through the Golgi apparatus.

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

Cancer cells educate natural killer cells to a metastasis-promoting cell state

2020-07-09T00:00

Chan et al. show that natural killer (NK) cells can be reprogrammed by breast cancer cells to promote metastasis. Reprogramming can be blocked by targeting NK cell inhibitory receptors TIGIT or KLRG1 or inhibiting DNA methyltransferases, which suggests new approaches to prevent or treat metastasis.

Natural killer (NK) cells have potent antitumor and antimetastatic activity. It is incompletely understood how cancer cells escape NK cell surveillance. Using ex vivo and in vivo models of metastasis, we establish that keratin-14⁺ breast cancer cells are vulnerable to NK cells. We then discovered that exposure to cancer cells causes NK cells to lose their cytotoxic ability and promote metastatic outgrowth. Gene expression comparisons revealed that healthy NK cells have an active NK cell molecular phenotype, whereas tumor-exposed (teNK) cells resemble resting NK cells. Receptor–ligand analysis between teNK cells and tumor cells revealed multiple potential targets. We next showed that treatment with antibodies targeting TIGIT, antibodies targeting KLRG1, or small-molecule inhibitors of DNA methyltransferases (DMNT) each reduced colony formation. Combinations of DNMT inhibitors with anti-TIGIT or anti-KLRG1 antibodies further reduced metastatic potential. We propose that NK-directed therapies targeting these pathways would be effective in the adjuvant setting to prevent metastatic recurrence.

PTPN21/Pez Is a Novel and Evolutionarily Conserved Key Regulator of Inflammation In Vivo

Drosophila provides a powerful model in which to study inflammation in vivo, and previous studies have revealed many of the key signaling events critical for recruitment of immune cells to tissue damage. In the fly, wounding stimulates the rapid production of hydrogen peroxide (H₂O₂).¹^,² This then acts as an activation signal by triggering a signaling pathway within responding macrophages by directly activating the Src family kinase (SFK) Src42A,³ which in turn phosphorylates the damage receptor Draper. Activated Draper then guides macrophages to the wound through the detection of an as-yet unidentified chemoattractant.3, 4, 5 Similar H₂O₂-activated signaling pathways are also critical for leukocyte recruitment following wounding in larval zebrafish,6, 7, 8, 9 where H₂O₂ activates the SFK Lyn to drive neutrophil chemotaxis. In this study, we combine proteomics, live imaging, and genetics in the fly to identify a novel regulator of inflammation in vivo; the PTP-type phosphatase Pez. Pez is expressed in macrophages and is critical for their efficient migration to wounds. Pez functions within activated macrophages downstream of damage-induced H₂O₂ and operates, via its band 4.1 ezrin, radixin, and moesin (FERM) domain, together with Src42A and Draper to ensure effective inflammatory cell recruitment to wounds. We show that this key role is conserved in vertebrates, because “crispant” zebrafish larvae of the Draper ortholog (MEGF10) or the Pez ortholog (PTPN21) exhibit a failure in leukocyte recruitment to wounds. This study demonstrates evolutionary conservation of inflammatory signaling and identifies MEGF10 and PTPN21 as potential therapeutic targets for the treatment of inflammatory disorders.

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Pez is a novel regulator of inflammation needed for macrophage recruitment to wounds

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During Drosophila inflammation, Pez acts in the H₂O₂/Src42a/Draper signaling axis

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Pez and Draper form dynamic clusters within macrophages in response to tissue damage

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Pez (PTPN21) and Draper (MEGF10) orthologs play conserved roles in fish leukocytes

Through the combination of proteomics, genetics, and live imaging, Campbell et al. identify Pez as a novel regulator of inflammation in vivo. Pez acts in the damage-sensing, H₂O₂/Src42a/Draper signaling axis, wherein it dynamically clusters with Draper to enable rapid leukocyte migration to epithelial wounds in both the fly and fish.

When Natural Behavior Engages Working Memory

Working memory (WM) enables temporary storage and manipulation of information,¹ supporting tasks that require bridging between perception and subsequent behavior. Its properties, such as its capacity, have been thoroughly investigated in highly controlled laboratory tasks.1, 2, 3, 4, 5, 6, 7, 8 Much less is known about the utilization and properties of WM in natural behavior,9, 10, 11 when reliance on WM emerges as a natural consequence of interactions with the environment. We measured the trade-off between reliance on WM and gathering information externally during immersive behavior in an adapted object-copying task.¹² By manipulating the locomotive demands required for task completion, we could investigate whether and how WM utilization changed as gathering information from the environment became more effortful. Reliance on WM was lower than WM capacity measures in typical laboratory tasks. A clear trade-off also occurred. As sampling information from the environment required increasing locomotion and time investment, participants relied more on their WM representations. This reliance on WM increased in a shallow and linear fashion and was associated with longer encoding durations. Participants’ avoidance of WM usage showcases a fundamental dependence on external information during ecological behavior, even if the potentially storable information is well within the capacity of the cognitive system. These foundational findings highlight the importance of using immersive tasks to understand how cognitive processes unfold within natural behavior. Our novel VR approach effectively combines the ecological validity, experimental rigor, and sensitive measures required to investigate the interplay between memory and perception in immersive behavior.

Video Abstract

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Gaze provides a measure of working-memory (WM) usage during natural behavior

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Natural reliance on WM is low even when searching for objects externally is effortful

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WM utilization increases linearly as searching for objects requires more locomotion

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The trade-off between using WM versus external sampling affects performance

Draschkow et al. demonstrate that reliance on working memory is low during natural behavior, even when “holding information in mind to guide future behavior” is the very essence of the task. As searching for objects externally increases in locomotive effort, WM usage increases in a shallow and linear fashion. This trade-off affects performance.

A Causal Role for Gastric Rhythm in Human Disgust Avoidance

Rotten food, maggots, bodily waste—all elicit disgust in humans. Disgust promotes survival by encouraging avoidance of disease vectors¹ but is also implicated in prejudice toward minority groups; avoidance of environmentally beneficial foods, such as insect protein; and maladaptive avoidance behavior in neuropsychiatric conditions.2, 3, 4, 5 Unlike fear, pathological disgust is not improved substantially by exposure therapy clinically,⁶ nor in experimental work does behavioral avoidance of disgusting images habituate following prolonged exposure.⁷^,⁸ Under normal physiological conditions, perception of disgusting stimuli disrupts myoelectrical rhythms in the stomach,9, 10, 11, 12, 13 inducing gastric dysrhythmias that correlate with neural signatures of disgust.¹¹ However, the causal role of gastric rhythm in disgust avoidance is unknown. We manipulated gastric rhythm using domperidone, a peripheral dopamine D2/D3 antagonist and common anti-emetic, at a dose (10 mg) that acts to convert gastric dysrhythmias to normal rhythms.⁹ In a preregistered, randomized, double-blind, placebo-controlled crossover design in 25 healthy volunteers (aged 18–25), we measured the effects of domperidone on core disgust avoidance, using eye tracking to measure implicit (oculomotor) avoidance of disgusting images (feces) before and after an “exposure” intervention (monetary reinforcement for looking at disgusting images).⁷^,⁸ We find that domperidone significantly reduces oculomotor disgust avoidance following incentivized exposure. This suggests that domperidone may weaken the “immunity” of disgust to habituation, putatively by reducing gastric dysrhythmias during incentivized engagement with disgusting stimuli. This indicates a causal role for disgust-related visceral changes in disgust avoidance, supporting the hypothesis that physiological homeostasis contributes to emotional experience.

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Healthy volunteers were given domperidone to normalize gastric rhythm or placebo

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We measured the effect of an exposure paradigm on oculomotor disgust avoidance

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Domperidone reduced oculomotor disgust avoidance after the exposure intervention

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This suggests gastric rhythm has a causal role in disgust avoidance

It is unknown whether gastric dysrhythmias contribute to disgust avoidance. By normalizing gastric state with domperidone and measuring oculomotor avoidance of disgusting images, Nord et al. discover that domperidone reduces oculomotor disgust avoidance following an exposure task. This suggests that gastric rhythm is one cause of disgust avoidance.

Calcineurin-dependent regulation of endocytosis by a plasma membrane ubiquitin ligase adaptor, Rcr1

2020-05-18T00:00

Rsp5 is an E3 ubiquitin ligase of the Nedd4 family that regulates many cellular processes in yeast. Zhu et al. show that two paralogous Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct locations. Exomer sorts Rcr1 to the plasma membrane via a new sorting motif, and the upregulation of Rcr1 via the calcineurin/Crz1 signaling pathway maintains cell integrity.

Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.

The deubiquitylase USP9X controls ribosomal stalling

2021-01-28T00:00

Clancy et al. develop a specific chemical inhibitor of USP9X and characterize its effects upon the cellular proteome. This analysis reveals a central role in the regulation of ribosomal stalling through control of critical E3 ligases.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

2020-05-20T00:00

To segregate chromosomes, the mammalian spindle must generate and respond to force. How it does so remains poorly understood. Pulling on the spindle using microneedles, Long et al. show that it can locally dissipate sustained force by regulating microtubule dynamics and breakage, thereby preserving global spindle structure.

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2020-06-18T00:00

The small Rho GTPase Cdc42 is a well-known regulator of cytoskeletal rearrangement and polarity development in all eukaryotic cell types. Here, Lu and Drubin report the serendipitous discovery of a novel Cdc42-ESCRT–nuclear envelope/endoplasmic reticulum connection.

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.

Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage

2020-06-29T00:00

Raso et al. find that high levels of interferon-stimulated gene 15 (ISG15), which is very frequent in cancer and robustly induced by pathogen infection, accelerate DNA replication fork progression, impacting genome stability and response to chemotherapy.

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-β, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

Induction of spontaneous human neocentromere formation and long-term maturation

2021-01-14T00:00

Murillo-Pineda et al. report a chromosome engineering system for human neocentromere formation and characterize the first experimentally generated human neocentromere. Neocentromere formation promotes local H3K9me3 eviction and cohesin and RNA polymerase II recruitment. Long-term culture results in gradual maturation of the inner centromere.

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Sas4 links basal bodies to cell division via Hippo signaling

2020-05-20T00:00

Sas4 is a conserved basal body assembly protein. Here, Ruehle et al. describe a previously unknown link between basal bodies and the control of cell division by Hippo signaling molecules that depends on Sas4.

Basal bodies (BBs) are macromolecular complexes required for the formation and cortical positioning of cilia. Both BB assembly and DNA replication are tightly coordinated with the cell cycle to ensure their accurate segregation and propagation to daughter cells, but the mechanisms ensuring coordination are unclear. The Tetrahymena Sas4/CPAP protein is enriched at assembling BBs, localizing to the core BB structure and to the base of BB-appendage microtubules and striated fiber. Sas4 is necessary for BB assembly and cortical microtubule organization, and Sas4 loss disrupts cell division furrow positioning and DNA segregation. The Hippo signaling pathway is known to regulate cell division furrow position, and Hippo molecules localize to BBs and BB-appendages. We find that Sas4 loss disrupts localization of the Hippo activator, Mob1, suggesting that Sas4 mediates Hippo activity by promoting scaffolds for Mob1 localization to the cell cortex. Thus, Sas4 links BBs with an ancient signaling pathway known to promote the accurate and symmetric segregation of the genome.

TANGLED1 mediates microtubule interactions that may promote division plane positioning in maize

2020-06-22T00:00

TAN1 is a microtubule-binding protein required for the spatial control of plant division plane orientation. TAN1 mediates both lateral and end-on microtubule interactions in vitro. These activities may promote proper division plane orientation in vivo.

The microtubule cytoskeleton serves as a dynamic structural framework for mitosis in eukaryotic cells. TANGLED1 (TAN1) is a microtubule-binding protein that localizes to the division site and mitotic microtubules and plays a critical role in division plane orientation in plants. Here, in vitro experiments demonstrate that TAN1 directly binds microtubules, mediating microtubule zippering or end-on microtubule interactions, depending on their contact angle. Maize tan1 mutant cells improperly position the preprophase band (PPB), which predicts the future division site. However, cell shape–based modeling indicates that PPB positioning defects are likely a consequence of abnormal cell shapes and not due to TAN1 absence. In telophase, colocalization of growing microtubules ends from the phragmoplast with TAN1 at the division site suggests that TAN1 interacts with microtubule tips end-on. Together, our results suggest that TAN1 contributes to microtubule organization to ensure proper division plane orientation.

CEP164C regulates flagellum length in stable flagella

2020-11-09T00:00

Stable eukaryotic flagella can maintain their length, and many protists grow new flagella while maintaining existing flagella. Mechanisms regulating this are not well understood. Atkins et al. show that CEP164C is important for this regulation, and these findings provide key novel insights into this process.

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.

A PHABULOSA-Controlled Genetic Pathway Regulates Ground Tissue Patterning in the Arabidopsis Root

In both animals and plants, development involves anatomical modifications. In the root of Arabidopsis thaliana, maturation of the ground tissue (GT)—a tissue comprising all cells between epidermal and vascular ones—is a paradigmatic example of these modifications, as it generates an additional tissue layer, the middle cortex (MC).1, 2, 3, 4 In early post-embryonic phases, the Arabidopsis root GT is composed of one layer of endodermis and one of cortex. A second cortex layer, the MC, is generated by asymmetric cell divisions in about 80% of Arabidopsis primary roots, in a time window spanning from 7 to 14 days post-germination (dpg). The cell cycle regulator CYCLIN D6;1 (CYCD6;1) plays a central role in this process, as its accumulation in the endodermis triggers the formation of MC.⁵ The phytohormone gibberellin (GA) is a key regulator of the timing of MC formation, as alterations in its signaling and homeostasis result in precocious endodermal asymmetric cell divisions.³^,⁶^,⁷ However, little is known on how GAs are regulated during GT maturation. Here, we show that the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) is a master regulator of MC formation, controlling the accumulation of CYCD6;1 in the endodermis in a cell non-autonomous manner. We show that PHB activates the GA catabolic gene GIBBERELLIN 2 OXIDASE 2 (GA2ox2) in the vascular tissue, thus regulating the stability of the DELLA protein GIBBERELLIN INSENSITIVE (GAI)—a GA signaling repressor—in the root and, hence, CYCD6;1 expression in the endodermis.

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PHB regulates cell non-autonomously the timing of MC formation

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A time-dependent rise of PHB expression controls the CYCD6;1 switch in the GT

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PHB regulates GAI stability modulating GA levels

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PHB regulates root GA levels activating GA2ox2 expression in the vasculature

Bertolotti, Unterholzner et al. show that a time-dependent threshold of the transcription factor PHB governs the timing of middle cortex formation in the Arabidopsis root. PHB promotes CYCD6;1 expression in the endodermis cell non-autonomously by reducing gibberellin (GA) levels in the vascular tissue and, hence, stabilizing the GAs repressor GAI.

Fission yeast Pak1 phosphorylates anillin-like Mid1 for spatial control of cytokinesis

2020-05-18T00:00

Magliozzi et al. show that fission yeast cell polarity kinase Pak1 regulates cytokinesis. Through a phosphoproteomic screen and subsequent mutant analysis, their work uncovers direct targets and mechanisms for Pak1 activity during cell division.

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place.

Host genetic effects in pneumonia

2020-12-13T00:00

Given the coronavirus disease 2019 (COVID-19) pandemic, investigations into host susceptibility to infectious diseases and downstream sequelae have never been more relevant. Pneumonia is a lung disease that can cause respiratory failure and hypoxia and is a common complication of infectious diseases, including COVID-19. Few genome-wide association studies (GWASs) of host susceptibility and severity of pneumonia have been conducted. We performed GWASs of pneumonia susceptibility and severity in the Vanderbilt University biobank (BioVU) with linked electronic health records (EHRs), including Illumina Expanded Multi-Ethnic Global Array (MEGA^EX)-genotyped European ancestry (EA, n= 69,819) and African ancestry (AA, n = 15,603) individuals. Two regions of large effect were identified: the CFTR locus in EA (rs113827944; OR = 1.84, p value = 1.2 × 10⁻³⁶) and HBB in AA (rs334 [p.Glu7Val]; OR = 1.63, p value = 3.5 × 10⁻¹³). Mutations in these genes cause cystic fibrosis (CF) and sickle cell disease (SCD), respectively. After removing individuals diagnosed with CF and SCD, we assessed heterozygosity effects at our lead variants. Further GWASs after removing individuals with CF uncovered an additional association in R3HCC1L (rs10786398; OR = 1.22, p value = 3.5 × 10⁻⁸), which was replicated in two independent datasets: UK Biobank (n = 459,741) and 7,985 non-overlapping BioVU subjects, who are genotyped on arrays other than MEGA^EX. This variant was also validated in GWASs of COVID-19 hospitalization and lung function. Our results highlight the importance of the host genome in infectious disease susceptibility and severity and offer crucial insight into genetic effects that could potentially influence severity of COVID-19 sequelae.

Auxin-Regulated Reversible Inhibition of TMK1 Signaling by MAKR2 Modulates the Dynamics of Root Gravitropism

Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.¹ Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1, 2, 3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1, 2, 3, 4 In particular, the timing of this dynamic response is regulated by PIN2,⁵^,⁶ but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).²^,7, 8, 9, 10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.

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MAKR2 is co-expressed with PIN2 and regulates the pace of root gravitropism

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MAKR2 controls PIN2 asymmetric accumulation at the root level during gravitropism

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MAKR2 binds to and is a negative regulator of the TMK1 receptor kinase

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Auxin antagonizes the MAKR2 inhibition of TMK1 by delocalizing MAKR2 in the cytosol

Marquès-Bueno, Armengot et al. show that the unstructured protein MAKR2 controls the dynamics of the root gravitropic response by acting as a negative regulator of the TMK1 receptor kinase. In addition, the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner.

Phasic Activation of Dorsal Raphe Serotonergic Neurons Increases Pupil Size

Transient variations in pupil size (PS) under constant luminance are coupled to rapid changes in arousal state,1, 2, 3 which have been interpreted as vigilance,⁴ salience,⁵ or a surprise signal.6, 7, 8 Neural control of such fluctuations presumably involves multiple brain regions⁵^,9, 10, 11 and neuromodulatory systems,³^,¹²^,¹³ but it is often associated with phasic activity of the noradrenergic system.⁹^,¹²^,¹⁴^,¹⁵ Serotonin (5-HT), a neuromodulator also implicated in aspects of arousal¹⁶ such as sleep-wake transitions,¹⁷ motivational state regulation,¹⁸ and signaling of unexpected events,¹⁹ seems to affect PS,20, 21, 22, 23, 24 but these effects have not been investigated in detail. Here we show that phasic 5-HT neuron stimulation causes transient PS changes. We used optogenetic activation of 5-HT neurons in the dorsal raphe nucleus (DRN) of head-fixed mice performing a foraging task. 5-HT-driven modulations of PS were maintained throughout the photostimulation period and sustained for a few seconds after the end of stimulation. We found no evidence that the increase in PS with activation of 5-HT neurons resulted from interactions of photostimulation with behavioral variables, such as locomotion or licking. Furthermore, we observed that the effect of 5-HT on PS depended on the level of environmental uncertainty, consistent with the idea that 5-HT could report a surprise signal.¹⁹ These results advance our understanding of the neuromodulatory control of PS, revealing a tight relationship between phasic activation of 5-HT neurons and changes in PS.

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Phasic optogenetic activation of DRN 5-HT neurons increases pupil size

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The effects of 5-HT neuron activation are not specific to behavioral states

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The effects of 5-HT on pupil size are linked to the level of uncertainty

The effects of serotonin (5-HT) on pupil size have not been investigated in detail. Cazettes et al. show that phasic optogenetic activation of 5-HT neurons causes pupil size changes in mice performing a foraging task. The 5-HT effects on pupil size are not specific to behavioral variables but appear to be linked to the task statistics.

Genomes of Pleistocene Siberian Wolves Uncover Multiple Extinct Wolf Lineages

Extant Canis lupus genetic diversity can be grouped into three phylogenetically distinct clades: Eurasian and American wolves and domestic dogs.¹ Genetic studies have suggested these groups trace their origins to a wolf population that expanded during the last glacial maximum (LGM)1, 2, 3 and replaced local wolf populations.⁴ Moreover, ancient genomes from the Yana basin and the Taimyr peninsula provided evidence of at least one extinct wolf lineage that dwelled in Siberia during the Pleistocene.³ ⁵ Previous studies have suggested that Pleistocene Siberian canids can be classified into two groups based on cranial morphology. Wolves in the first group are most similar to present-day populations, although those in the second group possess intermediate features between dogs and wolves.⁶ ⁷ However, whether this morphological classification represents distinct genetic groups remains unknown. To investigate this question and the relationships between Pleistocene canids, present-day wolves, and dogs, we resequenced the genomes of four Pleistocene canids from Northeast Siberia dated between >50 and 14 ka old, including samples from the two morphological categories. We found these specimens cluster with the two previously sequenced Pleistocene wolves, which are genetically more similar to Eurasian wolves. Our results show that, though the four specimens represent extinct wolf lineages, they do not form a monophyletic group. Instead, each Pleistocene Siberian canid branched off the lineage that gave rise to present-day wolves and dogs. Finally, our results suggest the two previously described morphological groups could represent independent lineages similarly related to present-day wolves and dogs.

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Pleistocene Siberian wolves represent multiple extinct evolutionary lineages

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Pleistocene wolves share ancestry with arctic dogs and some East Asian wolves

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A Paleolithic dog specimen is genetically similar to other Pleistocene wolves

Ramos-Madrigal et al. sequence the genomes of four Pleistocene Siberian wolves, two of which have divergent cranial morphologies. These canids represent multiple extinct lineages that dwelled in Siberia >50 ka ago and at least until 14.1 ka ago and that contributed to the genetic ancestry of arctic dogs and some East Asian wolves.

Immunodynamics of explanted human tumors for immuno‐oncology

2020-12-29T00:00

Decision making in immuno‐oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno‐assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti‐PD‐1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay.

To predict cancer resistance to PD‐1 blockade and design suitable combinations of immunomodulators, a 60‐h functional in sitro assay was set up in 43 tumors that allowed calculation of the “Immune Reactivity Score (IRS)” based on 17 TCR‐dependent‐ cytokines/chemokines.

Human soluble ACE2 improves the effect of remdesivir in SARS‐CoV‐2 infection

2020-12-14T00:00

There is a critical need for safe and effective drugs for COVID‐19. Only remdesivir has received authorization for COVID‐19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS‐CoV‐2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS‐CoV‐2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS‐CoV‐2 in both models. By using single amino‐acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub‐toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID‐19 clinical trials.

A human kidney organoid model was used to test antiviral drugs against SARS‐CoV‐2 infections, highlighting the efficiency of combining two different approaches to reduce SARS‐CoV‐2 viral load. Our findings open a promising way for clinical trials using safer and more efficient combination therapies in COVID‐19.

SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56

2020-12-31T00:00

The synaptonemal complex promotes faithful exchanges between homologous chromosomes during meiosis. Barbosa et al show that SCF-FBxo42 is important for assembly and maintenance of the synaptonemal complex by restricting the phosphatase PP2A-B56 level.

Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. Here, we report how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.

Phosphoregulation provides specificity to biomolecular condensates in the cell cycle and cell polarity

2020-05-12T00:00

Cells must control the composition and location of biomolecular condensates. This study shows that phosphorylation of the RNA-binding protein Whi3 is used to regulate functionally distinct condensates important for cell polarity and nuclear division in multinucleate fungus Ashbya gossypii.

Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.

Structural insights into G domain dimerization and pathogenic mutation of OPA1

2020-05-07T00:00

OPA1 mediates inner mitochondrial fusion and mutations cause optic atrophy. Yu et al. present the crystal structure of a minimal GTPase domain of human OPA1, which sheds light on its dimerization that is physiologically important and provides an explanation for pathogenic mutations.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF₃⁻, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

TORC1 inactivation stimulates autophagy of nucleoporin and nuclear pore complexes

2020-05-26T00:00

Autophagy selectively degrades a wide range of cellular components to regulate cellular functions or maintain cellular homeostasis. Tomioka et al. reveal that the nuclear pore complex and nucleoporins are degraded by selective autophagy upon inactivation of Tor kinase complex 1 in Saccharomyces cerevisiae.

The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor–binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope–derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term “NPC-phagy” and “nucleoporinophagy.”