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Biology subjects

Yu, C.-H.

Publications and source records attributed to Yu, C.-H..

At least 19 recordsLinked to original sources

Single Amino Acid Disease Mutations Act as Genetic Glues to Drive Neo-substrate Degradation

Disease-associated missense mutations are well known to affect protein turnover by either impairing substrate-ubiquitin ligase interactions or inducing misfolding-triggered protein clearance. Whether such mutations can drive gain-of-function in substrate-selective protein degradation remains under-explored. Here, we develop a tri-color global protein stability system to profile mutational effects across the intrinsically disordered regions (IDRs) of human transcription factors. Unexpectedly, we identify single amino acid substitutions that create functional short linear motif degrons to drive targeted protein degradation independently of protein quality control. These disease variants are exemplified by the gain of an FRY-like degron in GATA2, whose deficiency leads to impaired immune function. Structural analyses reveal that the substituted residue enables neomorphic interaction between the transcription factor and the KLHL15 E3 ligase by complementing their otherwise suboptimal interface. The prevalence of such glue-like mutations beyond transcription factors suggests that quasi-degrons are widespread in protein IDRs and can be functionalized by simple mutations, a finding that may inform the rational discovery of molecular glue degraders.

molecular biology↗

A Distinct Layer 1 Astrocyte Program Shapes Perisynaptic Structure and Calcium Signaling in Mouse Motor Cortex

Although layer-specific molecular and morphological diversity among cortical astrocytes is increasingly well established, how these distinct states are linked to specialized calcium signaling and maintained in the adult cortex remains unclear. Here, combining super-resolution structural imaging, two-photon calcium imaging, and analysis of public single-cell and spatial transcriptomic resources, we identify Layer 1 (L1) astrocytes in mouse primary motor cortex as a distinct superficial astrocyte program. These cells occupy compact territories yet contain dense synapse-associated loop-like structures and display frequent, fast, broadly spreading calcium events that engage a large fraction of the territory. Transcriptomic analyses identify Id1 and Id3 as enriched components of this superficial program. CRISPR-Cas9 deletion of Id1 and Id3 in adult astrocytes selectively disrupts L1 and superficial Layer 2/3 (L2/3) astrocytes, expanding territory size, reducing fine-process complexity, and suppressing calcium activity. Thus, adult layer-specific transcriptional programs maintain specialized astrocyte structure and signaling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/712002v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@89c916org.highwire.dtl.DTLVardef@1f0d71eorg.highwire.dtl.DTLVardef@bb02f3org.highwire.dtl.DTLVardef@1bedf7e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILayer 1 astrocytes in mouse motor cortex have compact, loop-rich nanoarchitecture C_LIO_LILayer 1 astrocytes show fast, frequent, widespread calcium events C_LIO_LISingle-cell data define a superficial astrocyte state enriched for Id1 and Id3 C_LIO_LIAdult Id1/Id3 loss selectively disrupts Layer 1 astrocyte structure and calcium C_LI In BriefBhattacharjee et al., identify a specialized Layer 1 astrocyte state in mouse motor cortex, defined by compact, loop-rich nanoarchitecture and unusually high calcium signaling. Id1/Id3 deletion selectively disrupts this superficial program, linking layer-specific astrocyte form and function to adult transcriptional control.

neuroscience↗

MEX3A control of mitochondrial fitness is essential for ovarian clear cell carcinoma tumorigenesis and liver metastasis

Ovarian cancer (OC) is highly metastatic and chemoresistant. Due to heterogeneity among OC subtypes, the mechanisms underlying OC malignancy and metastasis remain largely unknown. Ovarian clear cell carcinoma (OCCC) accounts for 5-25% of OC and its incidence rate is rising. Liver metastasis is particularly high in OCCC patients and leads to significantly reduced median survival. Why OCCC metastasizes to liver at such high frequency remains elusive. We previously identified MEX3A as a key factor that promotes OCCC tumorigenesis in part by circumventing p53-mediated ferroptosis. Here, we report that MEX3A control of mitochondrial fitness, which occurs independently of p53, is essential for OCCC primary tumor growth and liver metastasis. MEX3A depletion resulted in chronic mitochondrial fragmentation and accumulation of non-functional mitochondria. MEX3A-depleted cells had decreased mitochondrial membrane potential, increased superoxide and decreased NAD+/NADH ratio, resulting in inhibition of oxidative phosphorylation (OXPHOS) and decreased ATP levels. In an environment enriched with mitophagy stressors, such as the liver, MEX3A-depleted OCCC cells had greatly reduced survival due to failure to recover from mitophagy. Consistent with these observations, MEX3A knockdown greatly reduced liver metastasis. Together, these data demonstrate that MEX3A-mediated mitochondrial fitness is a major factor underlying its p53-independent promotion of OCCC tumorigenesis and liver metastasis. Thus, targeting MEX3A will be a promising strategy to inhibit OCCC progression. Statement of significanceUnexpected effects of MEX3A on mitochondrial fitness indicate that the need for high MEX3A expression is an OCCC vulnerability that can be exploited to uncover new treatment strategies.

cancer biology↗

BMAL2 is a druggable target for ARID1A-wildtype ovarian clear cell carcinoma (OCCC)

Ovarian clear cell carcinoma (OCCC) is highly chemo-resistant and has worse clinical outcome at advanced stages than other ovarian cancer subtypes. The most frequent ([~]50%) alterations in OCCC are AT-rich interactive domain 1A gene (ARID1A) mutations which lead to ARID1A deficiency. However, OCCC that retains ARID1A function differs substantially from ARID1A mutated OCCC. Particularly, targeted therapies that sensitize ARID1A-deficient OCCC to DNA damage are largely ineffective against OCCC with wild-type (wt) ARID1A. Thus, it is important to identify druggable targets and develop targeted therapies specifically for ARID1A-wt OCCC. We identified BMAL2 as a critical OCCC oncogene that promotes tumorigenesis by preventing DNA damage from endogenous origins. BMAL2 depletion altered expression of genes in DNA damage repair pathways, including RAD51, a core enzyme of the homologous recombination (HR) pathway. This led to double-stranded break accumulation, decreased cell viability and reduced tumor growth. This dependence on BMAL2 to maintain DNA integrity and cell viability can be a new route to suppress ARID1A-wt OCCC. Consistent with this idea, we found that GW833972A, a cannabinoid receptor agonist, bound BMAL2 with high affinity and facilitated its degradation. This in turn reduced RAD51 expression, leading to an accumulation of DNA damage and decreased cell viability. Xenograft models further demonstrated that GW833972A treatment alone inhibited ARID1A-wt OCCC tumor growth. Together, our findings reveal an essential oncogenic role of BMAL2 and demonstrate that it is an appealing therapeutic target, especially for ARID1A-wt OCCC. Statement of significanceBMAL2 depletion, or degradation by a small molecule, led to DNA damage accumulation, decreased cell viability and reduced tumorigenesis of ARID1A-wildtype ovarian clear cell carcinoma, indicating that BMAL2 is an appealing therapeutic target for treating this malignant disease.

cancer biology↗

Optogenetic construction of de novo integrin-adhesion complexes reveals role for biocondensation in adhesion nucleation

Integrin-adhesion complexes (IACs) form spontaneously in cells on extracellular matrix substrates, allowing them to sense matrix composition and transduce force. However, IACs often do not form uniformly across a cell, which begs the question: What is required to nucleate an adhesion, and what factors enable the stabilization of an IAC once it has formed? Many factors have been suggested to promote formation and the subsequent stabilization of IACs. It is difficult to explicitly test these factors in vivo as IACs undergo constant remodeling. Here, we employ optogenetics to explicitly test the ability of talin in different activity and phase states to nucleate and stabilize IACs in regions where none are present. We find that fusion of intrinsically disordered regions directly to talin enhances its adhesion nucleation potential and allows new adhesions to be produced in response to optogenetic talin clustering. Similarly, expression of factors previously shown to enhance biocondensation in vitro, such as paxillin, the paxillin N-terminus, or unfolding of talin, allows for adhesion nucleation and biocondensation of talin. We show that these biocondensates of talin can cluster and activate integrins even in the absence of extracellular matrix. By applying optogenetic activation to regions of the cell with or without ventral actomyosin, we demonstrate actomyosin engagement promotes the formation and stability of adhesions. These results are corroborated by theoretical modelling which shows that phase separation of talin is enhanced by differential clutch formation in the presence of actomyosin thus enabling peripheral adhesion formation and stability. This work establishes a model in which increased cooperativity of talin enables IAC nucleation through talin biocondensation, which clusters and activates integrins. In addition to these findings, we generate multiple optogenetic tools that enable local nucleation or enhancement of IACs. HighlightsO_LIOptogenetic tools mediating talin biocondensation can locally induce focal adhesion formation C_LIO_LIPaxillin LD domains enable biocondensation of talin C_LIO_LIBiocondensation of talin enables IAC formation C_LIO_LIPhase separation of talin can activate integrins independently of ECM C_LIO_LIComputational model reconciles spatial variance in LLPS and IAC formation. C_LI

cell biology↗

Open-source modular FPGA system for two-photon mesoscope enabling multi-layer, multi-depth neural activity recording and lifetime imaging

Large field-of-view (FOV) two-photon microscopy makes it possible to record a large number of neural activities from multiple brain regions simultaneously. However, the larger the field of view, the longer it takes to scan the entire FOV. To increase imaging speed, we have developed open-source software to digitize analogue signals from a photomultiplier tube using a field-programmable gate array (FPGA) at a rate of 3.2 GS/s. By combining this with a newly developed a circular delay-path module for a custom two-photon mesoscope (Diesel2p), we succeeded in simultaneous recording of >10,000 neurons from the entire bilateral dorsal cortex at up to four depths. We also demonstrated large FOV lifetime imaging using the same system. Our modular, open-source FPGA system can be readily integrated into any type of two-photon microscope and will accelerate the biomedical application of multi-scale two-photon imaging in a wide range of pathophysiological investigations.

neuroscience↗

Non-inertial scan angle multiplier for expanded fields-of-view

In many laser scan engines, mechanical inertia imposes a tradeoff between mirror diameter (mass) and scan speed (cycle time) and/or scan angle amplitude. Ultimately, this limits the field-of-view and/or scan speed in multiphoton and confocal imaging, laser-based manufacturing, and other applications. To push parameters past this inertia limit, we present a non-inertial (stationary) add-on unit that doubles the scan angle amplitude while preserving both the beam size and cycle time, thus doubling the etendue (optical invariant) and overall system throughput. We demonstrate its use in two-photon calcium imaging of neural activity in living mice. We also adapt the approach to present a phase doubling unit that doubles the maximum range of phase modulators. Finally, we describe further variants including generalizing the approach to Nx scan angle multiplication (N = 2, 3, 4, ...) with diffraction limited performance across a wide scan angle range and over a broad wavelength bandwidth. These non-inertial scan angle multipliers (called Nisam2x, 3x, ...) can expand the capabilities of a range of technologies including imaging, adaptive optics, sensing, marking, and manufacturing.

neuroscience↗

Abnormal synaptic proteomes, impaired neural ensembles, and defective behaviors in autism mouse models are ameliorated by dietary intervention with nutrient mixtures

Autism spectrum disorders (ASD) are a group of heterogeneous, behaviorally defined neurodevelopmental conditions influenced by both genetic and environmental factors. Here, we show that nutrients--an important environmental factor--can modulate synaptic proteomes, reconfigure neural ensembles, and improve social behaviors in ASD mouse genetic models. We analyzed Tbr1+/- mice, a well-established model of ASD, using proteomic approaches and in vivo calcium imaging. Synaptic and metabolic proteomes were found to be sensitive to Tbr1 haploinsufficiency. Our results also revealed that Tbr1 haploinsufficiency promotes hyperactivation and hyperconnectivity of basolateral amygdala (BLA) neurons, enhancing the activity correlation between individual neurons and their corresponding ensembles. Zinc, branched-chain amino acids (BCAA), and serine--all nutrients known to regulate synapse formation and activity--were then combined into supplement cocktails and administered to Tbr1+/- mice. This treatment altered synaptic and metabolic proteomes and normalized the activity and connectivity of the BLA in Tbr1+/- mice during social interactions. We further show that although a low dose of individual nutrients did not alter social behaviors, treatment with supplement cocktails containing low-dose individual nutrients improved social behaviors and associative memory of Tbr1+/- mice, implying a synergistic effect of combining low-dose zinc, BCAA, and serine. Moreover, the supplement cocktails also improved social behaviors in Nf1+/- and Cttnbp2+/M120I mice, two additional ASD mouse models. Thus, our findings reveal aberrant neural connectivity in the BLA of Tbr1+/- mice and indicate that dietary supplementation with zinc, BCAA, and/or serine offers a safe and accessible approach to mitigate neural connectivity and social behaviors across multiple ASD models.

neuroscience↗

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

neuroscience↗

Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans

Recent studies have highlighted the significance of the spindle midzone, the region between the segregating chromosomes, in ensuring proper chromosome segregation. By combining 3D electron tomography, cutting-edge light microscopy and a novel single cell in vitro essay allowing single molecule tracking, we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans by the chromokinesin KLP-19, and its relevance for proper spindle function. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and KLP-19 in C. elegans, the homologs of PRC1 and KIF4a, suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length and thus microtubule-based interactions in the spindle midzone, which affects spindle dynamics and force transmission. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone. SummaryKLP-19 controls microtubule length in the spindle midzone of C. elegans, affecting spindle dynamics and force transmission during mitosis.

cell biology↗

Mesoscale functional architecture in medial posterior parietal cortex

The posterior parietal cortex (PPC) in mice has various functions including multisensory integration1-3, vision-guided behaviors4-6, working memory7-13, and posture control14,15. However, an integrated understanding of these functions and their cortical localizations in and around the PPC and higher visual areas (HVAs) has not been completely elucidated. Here we simultaneously imaged the activity of thousands of neurons within a 3 x 3 mm2 field-of-view, including eight cortical areas around the PPC, during behavior with a two-photon mesoscope16. Mice performed both a vision-guided task and a choice history-dependent task, and the imaging results revealed distinct, localized, behavior-related functions of two medial PPC areas. Neurons in the anteromedial (AM) HVA responded to both vision and choice information, and thus AM is a locus of association between these channels. By contrast, the anterior (A) HVA stores choice history with sequential dynamics and represents posture. Mesoscale correlation analysis on the intertrial variability of neuronal activity demonstrated that neurons in AM exhibited diverse, area-dependent interactions, while neurons in area A shared fluctuations with the primary somatosensory area. Pairwise interareal interactions among neurons were precisely predicted by the anatomical input correlations, with the exception of some global interactions. Thus, the medial PPC has two distinct modules, areas AM and A, with each having distinctive modes of cortical communication. These medial PPC modules can serve separate higher-order functions: area AM for multisensory and cognitive integration with locally processed signals and area A for transmission of information including posture, movement, and working memory.

neuroscience↗

The Cousa objective: a long working distance air objective for multiphoton imaging in vivo

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue, but applying this technique in vivo can be limited by short working distance water-immersion objectives. Here we present an ultra long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a > 4 mm2 field-of-view with submicron lateral resolution, and is compatible with commonly used multiphoton imaging systems. We share the full optical prescription, and report performance including in vivo 2-photon and 3-photon imaging in a range of species and preparations.

neuroscience↗

Long-term Transverse Imaging of the Hippocampus with Glass Microperiscopes

The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.

neuroscience↗

Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large Field-of-view Two-Photon imaging (Diesel2p). Combining novel optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view ([~] 25 mm2) with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.

neuroscience↗

Microtubule re-organization during female meiosis in C. elegans

The female meiotic spindles of most animals are acentrosomal and undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of acentrosomal spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic C. elegans spindles undergoing the transition from metaphase to anaphase. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over on second time scales. The results show that the transition from metaphase to anaphase correlates with an increase in the number of microtubules and a decrease in their average length. Detailed analysis of the tomographic data revealed that the length of microtubules changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for those microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the observed large-scale reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those that are closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe alone. In anaphase, even microtubules close to the chromosomes show no signs of cutting. This suggests that the most prominent drivers of spindle rearrangements from metaphase to anaphase are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on the presence of katanin.

cell biology↗

Cryptococcus neoformans resist to drastic conditions by switching to viable but non-culturable cell phenotype

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce C. neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in Cryptococcus neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. All together, these data show that we were able to generate for the first time VBNC phenotype in Cryptococcocus neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

microbiology↗

Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central spindle microtubules during chromosome segregation in human mitotic spindles, and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central spindle microtubules during chromosome segregation in diverse spindles, and suggest that central spindle microtubules and chromosomes are strongly coupled in anaphase.

cell biology↗

Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Forster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

cell biology↗