Biology subjects

Cox, D.

Publications and source records attributed to Cox, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Four-dimensional quantitative analysis of cell plate development using lattice light sheet microscopy identifies robust transition points between growth phases

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodeling, and timely polysaccharide deposition, such as callose. Such a dynamic process requires dissection in time and space; hence this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM) we studied cell plate development in four dimensions, monitored by the behavior of the cytokinesis specific GTPase RABA2a. We monitored the entire length of cell plate development, from its first emergence, with the aid of RABA2a, both in the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis, we identified distinct behavioral patterns allowing for the identification of three easily trackable, cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited phase transition, establishing quantitatively the critical role and timing of the polysaccharide in cell plate expansion and maturation. This study exemplifies the power of LLSM, combined with quantitative analysis to decode and untangle such a complex process. HighlightWe employed lattice light sheet 4D microscopy in plants to dissect cytokinesis, a multistage process involving orchestrating delivery of membranes and timely polysaccharide deposition. Robust quantitative analysis revealed distinct phase shifts, while inhibition of callose deposition abolished the phase transition.

plant biology↗

bioRxiv2023

Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.

synthetic biology↗

bioRxiv2023

Characterization and super-resolution imaging of small tau aggregates in human samples.

Hyperphosphorylation and aggregation of the microtubule binding protein tau plays a key role in the development of Alzheimers disease. While the structure of the filamentous aggregates formed in humans has recently been determined to atomic resolution, there is far less information available about the smaller aggregate precursors, thought to be the most neurotoxic. To address this gap, we have developed a single molecule pull-down (SiMPull) able to detect tau aggregates in clinically relevant human samples. This method enables the detection and characterisation of individual tau aggregates, as opposed to averaged features obtained from traditional bulk techniques. We report the number, size and shape of individual aggregates measured via super-resolution microscopy, revealing disease-specific differences in tau aggregate morphology. By adapting the assay to simultaneously detect multiple phosphorylation sites in individual aggregates, we were also able to derive compositional profiles for pathological modifications present in individual aggregates. We demonstrate that tau aggregates in Alzheimers disease are significantly more likely to contain both the AT8 and T181 pathological phosphorylation markers, rather than only one. Together, tau SiMPull identified distinct subpopulations of large, modified tau aggregates that were invisible to traditional methodologies. These morphological and compositional differences distinguish samples taken from disease cohorts, offering to illuminate underlying disease mechanisms, and providing a foundation for novel diagnostic strategies.

biochemistry↗

bioRxiv2023

Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo (Hanna 2019). To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lung, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lung.

cancer biology↗

bioRxiv2022

Cellular aging impedes stress-activation of a crucial JDP-Hsp70 protein disaggregase

The collapse of protein homeostasis manifests itself in a toxic protein aggregation cascade, which is associated with degenerative diseases and aging. To solubilize aggregates, dedicated protein disaggregases exist in unicellular organisms, but these have no nuclear/cytosolic orthologs in metazoa. Alternative metazoan disaggregation machines have been described, but how these are operated and regulated in vivo remained unknown. We show that protein disaggregases are functionally diversified in human cells to efficiently target different types of stress-induced aggregates in sequential and temporally distinct phases. In particular, we show the selective assembly of an Hsp70-DNAJA1-DNAJB1 trimeric disaggregase that forms during late phase of stress recovery., i.e., after VCP-dependent solubilization of non-native proteins that accumulate in cellular condensates such as nucleoli or stress granules. When activated, the trimeric disaggregase provides resistance to stress toxicity and contributes to amyloid disposal. Strikingly, this disaggregase collapses early in cells undergoing replicative aging with important underlining pathophysiological consequences.

cell biology↗

bioRxiv2022

Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli.

Accurate spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. To address this need, protein painting methods have emerged as a way to gain insight into the conformational status of proteins within cells at the proteome-wide scale. For example, tetraphenylethene maleimide (TPE-MI) has previously been used to quantify the engagement of quality control machinery with client proteins in cell lysates. Here, we showcase the ability of TPE-MI to additionally reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to individual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.

cell biology↗

bioRxiv2021

ChAdOx1 nCoV-19 (AZD1222) vaccine elicits monoclonal antibodies with potent cross-neutralizing activity against SARS-CoV-2 viral variants

Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that despite a relatively low serum neutralization titre, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (B.1.1.7, P.1, B.1.351 and B.1.617.2) were obtained. The vaccine elicited neutralizing mAbs form 8 distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222 elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post infection. Spike reactive IgG+ B cells were still detectable 9-months post boost. These findings give molecular insights into AZD1222 elicited antibody response.

immunology↗

bioRxiv2021

Sequence grammar underlying unfolding and phase separation of globular proteins

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs. HighlightsO_LIUnfolded states of globular proteins phase separate to form UPODs in cells C_LIO_LIThe fraction of unfolded molecules and the sticker grammar govern phase separation C_LIO_LIHydrophobic residues act as stickers that engage in intermolecular interactions C_LIO_LISticker grammar also influences gain-of-function recruitment into aberrant UPODs C_LI

biophysics↗

bioRxiv2021

Virulence determinant, PTP7, controls vesicle budding from the Maurers clefts, adhesin protein trafficking and host cell remodeling in Plasmodium falciparum

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins P. falciparum malaria pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurers cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurers clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, found only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking. Author SummaryWe describe a malaria parasite protein involved in virulence factor trafficking (PTP7) that moves between different compartments in the host red blood cell cytoplasm in a stage-dependent manner. Upon disruption of the PTP7 locus, the Maurers cleft trafficking compartments become decorated with vesicles; the knobby protrusions on the host red blood cell surface are depleted and distorted; and trafficking of the virulence protein, EMP1, to the host red blood cell surface is ablated. We provide evidence that a region of PTP7 with low sequence complexity plays an important role in driving fission of vesicles from the Maurers clefts.

biochemistry↗

bioRxiv2021

A biosensor to gauge protein homeostasis resilience differences in the nucleus compared to cytosol of mammalian cells

An extensive network of chaperones and other proteins maintain protein homeostasis and guard against inappropriate protein aggregation that is a hallmark of neurodegenerative diseases. Using a fluorescence resonance energy-based biosensor that simultaneously reports on intact cellular chaperone holdase activity and detrimental aggregation propensity, we investigated the buffering capacity of the systems managing protein homeostasis in the nucleus of the human cell line HEK293 compared to the cytosol. We found that the nucleus showed lower net holdase activity and reduced capacity to suppress protein aggregation, suggesting that the nuclear quality control resources are less effective compared to those in the cytosol. Aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to deplete cytosolic chaperone supply by depleting holdase activity. Unexpectedly, the same stress increased holdase activity in the nucleus suggesting that proteostasis stress can trigger a rebalance of chaperone supply in different subcellular compartments. Collectively the findings suggest the cytosol has more capacity to manage imbalances in proteome foldedness than the nucleus, but chaperone supply can be redirected into the nucleus under conditions of proteostasis stress caused by cytosolic protein aggregation.

biochemistry↗

bioRxiv2021

Hidden information on protein function in censuses of proteome foldedness

Methods that assay protein foldedness with proteomics have generated censuses of protein folding stabilities in biological milieu. Surprisingly, different censuses poorly correlate with each other. Here, we show that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding. About one quarter of cysteine or methionine sidechains in proteins in mammalian cell lysate increase in reactivity upon chemical denaturant titration consistent with two-state unfolding. Paradoxically, up to one third decreased reactivity, which were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings. One Sentence SummaryWe show that proteome folding stability censuses are ill-defined because they earmark hidden information on conformation and ligand binding.

biochemistry↗

bioRxiv2020

Stable 3D Head Direction Signals in the Primary Visual Cortex

Visual signals influence the brains computation of spatial position and orientation. Accordingly, the primary visual cortex (V1) is extensively interconnected with areas involved in computing head direction (HD) information. Predictive coding theories posit that higher cortical areas send sensory or motor predictions to lower areas, but whether this includes cognitive variables like the HD signal--and whether HD information is present in V1--is unknown. Here we show that V1 encodes the yaw, roll, and pitch of the head in freely behaving rats, either in the presence or absence of visual cues. HD tuning was modulated by lighting and movement state, but was stable on a population level for over a week. These results demonstrate the presence of a critical spatial orientation signal in a primary cortical sensory area and support predictive coding theories of brain function.

neuroscience↗

bioRxiv2020

Computational model of neurodegenerative damage in the olfactory bulb: role of bulb and damage geometry, damage site, and identification of a potential non-invasive marker of early Parkinson's disease onset

Several neurodegenerative diseases impact the olfactory system, and in particular the olfactory bulb, early in disease progression. One mechanism by which damage occurs is via synaptic dysfunction. Here, we implement a computational model of the olfactory bulb and investigate the effect of weakened connection weights on network oscillatory behavior. Olfactory bulb network activity can be modeled by a system of equations that describes a set of coupled nonlinear oscillators. In this modeling framework, we propagate damage to synaptic weights using several strategies, varying from localized to global. Damage propagated in a dispersed or spreading manner leads to greater oscillatory power at moderate levels of damage. This increase arises from a higher average level of mitral cell activity due to a shift in the balance between excitation and inhibition. That this shift leads to greater oscillations critically depends on the nonlinearity of the activation function. Linearized analysis of the network dynamics predicts when this shift leads to loss of oscillatory activity. We thus demonstrate one potential mechanism involved in the increased gamma oscillations seen in some animal models of Alzheimers disease and highlight the potential that pathological olfactory bulb behavior presents as an early biomarker of disease.

neuroscience↗

bioRxiv2020

A Biophysical Model for Plant Cell Plate Development

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a cell plate that partitions the cytoplasm of the dividing cell. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to the form the nascent cell wall by the timely deposition of polysaccharides such as callose, cellulose, and crosslinking glycans. In contrast to the role of endomembrane protein regulators the role of polysaccharides, in cell plate development is poorly understood. Callose, a {beta}-1-3 glucan polymer, is transiently accumulated during cell plate expansion to be replaced by cellulose in mature stages. Based on the severity of cytokinesis defects in the absence of callose, it has been proposed that it stabilizes this membrane network structure. However, there is currently no theory to understand its role in cytokinesis. Here we extend the Helfrich free energy model for membranes including a phenomenological spreading force as an "areal pressure" generated by callose and/or other polysaccharides. Regular cell plate development in the model is possible, with suitable bending modulus, for a two-dimensional late stage spreading force parameter of between 2-6pN/nm, an osmotic pressure difference of 2-10kPa, and spontaneous curvature between 0-0.04nm-1. With these conditions, stable membrane conformation sizes and morphologies emerge in concordance with stages of cell plate development. With no spreading force, the cell plate fails to mature properly, corroborating experimental observations of cytokinesis arrest in the absence of callose. To reach a nearly mature cell plate, our model requires the late stage onset that the spreading force coupled with a concurrent loss of spontaneous curvature. A simple model based upon production of callose as a quasi-two-dimensional self-avoiding polymer produces the correct phenomenological form of the spreading force, which will be further refined, since matching to our numbers requires an exceptionally high callose synthesis rate. Significance StatementPlant cell division features the development of a unique membrane network called the cell plate that matures to a cell wall which separates the two daughter cells. During cell plate development, callose, a {beta}-1-3 glucan polymer, is transiently synthesized at the cell plate only to be replaced by cellulose in mature stages. The role for this transient callose accumulation at the cell plate is unknown. It has been suggested that callose provides mechanical stability, as well as a spreading force that widens and expands tubular and fenestrated cell plate structures to aid the maturation of the cell plate. Chemical inhibition of callose deposition results in the failure of cell plate development supporting this hypothesis. This publication establishes the need for a spreading force in cell plate development using a biophysical model that predicts cell plate development in the presence and the absence of this force. Such models can potentially be used to decipher for the transition/maturation of membrane networks upon the deposition of polysaccharide polymers.

biophysics↗

bioRxiv2020

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

Mutations that cause Huntingtons Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntingtons disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and examined whether ribosome quality control machinery previously implicated in stalled ribosomes were involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation.

cell biology↗

bioRxiv2020

Natural variation in autumn FLC levels, rather than epigenetic silencing, aligns vernalization to different climates

Plants monitor temperatures over long timescales to assess seasons and time developmental transitions. In Arabidopsis thaliana, winter is registered during vernalization through the temperature-dependent repression and epigenetic silencing of floral repressor FLOWERING LOCUS C (FLC). Natural Arabidopsis accessions show considerable variation in vernalization, however which aspect of the FLC repression mechanism is most important for adaptation to different climates is not clear. By analyzing FLC silencing in natural variants throughout winter in three field sites, we find that FLC starting levels and early phases of silencing are the major variables underlying vernalization response, rather than establishment of epigenetic silencing. This results in an intricate interplay between promotion and delay of flowering to balance survival, and through a post-vernalization effect of FLC, reproductive effort via branch production. These data reveal how non-coding FLC variation aligns vernalization response to different climatic conditions and year-on-year fluctuations in natural temperature profiles. Impact StatementAlleles of the major floral repressor vary in their initial expression to underpin the ability of Arabidopsis to survive year-on-year climatic fluctuations.

molecular biology↗

bioRxiv2020

Spatio-temporal mapping of mechanical force generated by macrophages during FcγR-dependent phagocytosis reveals adaptation to target stiffness

Macrophage phagocytosis is a strikingly flexible process central to pathogen clearance and is an attractive target for the development of anti-cancer immunotherapies. To harness the adaptability of phagocytosis, we must understand how macrophages can successfully deform their plasma membrane. While the signaling pathways and the molecular motors responsible for this deformation have been studied for many years, we only have limited insight into the mechanics that drive the formation of the phagocytic cup. Using Traction Force Microscopy (TFM), we have been able to characterize the spatio-temporal dynamics of mechanical forces generated in the course of Fc{gamma}R-dependent frustrated phagocytosis and we determined whether this was affected by the stiffness of the potential phagocytic targets. We observed that frustrated phagocytosis is an atypical form of spreading where the cell deformation rate is unaffected by the substrate stiffness. Interestingly, the cell initially extends without forces being recorded then switches to a mode of pseudopod extension involving spatially organized force transmission. Importantly we demonstrate that macrophages adapt to the substrate stiffness primarily through a modulation of the magnitude of mechanical stress exerted, and not through modification of the mechanical stress kinetics or distribution. Altogether, we suggest that macrophage phagocytosis exhibits a clear resilience to variations of the phagocytic target stiffness and this is favored by an adaptation of their mechanical response.

cell biology↗

bioRxiv2020

Astroglial FMRP modulates synaptic signaling and behavior phenotypes in FXS mouse model

Fragile X syndrome (FXS) is one of the most common inherited intellectual disability (ID) disorders, in which the loss of FMRP protein induces a range of cellular signaling changes primarily through excess protein synthesis. Although neuron-centered molecular and cellular events underlying FXS have been characterized, how different CNS cell types are involved in typical FXS synaptic signaling changes and behavioral phenotypes is largely unknown. Recent evidence suggests that selective loss of astroglial FMRP is able to dysregulate glutamate uptake, increase spine density, and impair motor-skill learning. Here we investigated the effect of astroglial FMRP on synaptic signaling and FXS-related behavioral and learning phenotypes in astroglial Fmr1 cKO and cON mice in which FMRP expression is selectively diminished or restored in astroglia. We found that selective loss of astroglial FMRP contributes to cortical hyperexcitability by enhancing NMDAR-mediated evoked but not spontaneous miniEPSCs and elongating cortical UP state duration. Selective loss of astroglial FMRP is also sufficient to increase locomotor hyperactivity, significantly diminish social novelty preference, and induce memory acquisition and extinction deficits in astroglial Fmr1 cKO mice. Importantly, re-expression of astroglial FMRP is able to significantly rescue the hyperactivity (evoked NMDAR response, UP state duration, and open field test) and social novelty preference in astroglial Fmr1 cON mice. These results demonstrate a profound role of astroglial FMRP in the evoked synaptic signaling, spontaneously occurring cortical UP states, and FXS-related behavioral and learning phenotypes and provide important new insights in the cell type consideration for the FMRP reactivation strategy.

neuroscience↗