Biology subjects

Wong, G. C. L.

Publications and source records attributed to Wong, G. C. L..

10 recordsLinked to original sources

bioRxiv2025

Drp1 Proteins Released from Hydrolysis-driven Scaffold Disassembly Trigger Nucleotide-dependent Membrane Remodeling to Promote Scission

Dynamin-related protein (Drp1) drives mitochondrial fission, dysregulation of which leads to neurodegenerative, metabolic, and apoptotic disorders. The precise mechanism of fission completion is unclear. One prevailing model is based on GTP-driven assembly of Drp1 helices that increase confinement via force generation. However, constriction to nanoscopic tubule radii appears necessary but not sufficient for scission. The other is based on GTP-driven disassembly of a constricting Drp1 scaffold that drives a membrane disturbance, but the relation of disassembly to scission and GTP hydrolysis remain uncertain. Elucidation of mitochondrial fission is complicated by the multiple time-involved in the dynamics of mechanoenzyme activity, oligomer disassembly, and membrane remodeling. Using machine learning, synchrotron x-ray scattering, and a theoretical model, our data support a model where progressive GTP hydrolysis enable free Drp1s to increase their capacity for inducing membrane negative Gaussian curvature (NGC). Furthermore, we identify and Drp1 variants that diminish this progressive capacity. Machine learning reveals that predicted NGC-generating sequences of the Drp1 oligomer are not in contact with the confined lipid tube, that scission-enhancing membrane remodeling is triggered by free Drp1 released upon disassembly. TeaserFree Drp1 released from disassembly of oligomeric Drp1 constrictase promotes GTP-hydrolysis dependent mitochondrial scission.

biophysics↗

bioRxiv2024

Dynamins combine mechano-constriction and membrane remodeling to enable two-step mitochondrial fission via a snap-through instability.

Mitochondrial fission is controlled by dynamin proteins, the dysregulation of which is correlated with diverse diseases. Fission dynamins are GTP hydrolysis-driven mechanoenzymes that self-oligomerize into helical structures that constrict membrane to achieve fission, but details are not well understood. However, dynamins can also remodel membranes by inducing negative Gaussian curvature, the type of curvature required for completion of fission. Here, we examine how these drastically different mechanisms synergistically exert their influences on a membrane, via a mechanical model calibrated with small-angle X-ray scattering structural data. We find that free dynamin can trigger a "snap-through instability" that enforces a shape transition from an oligomer-confined cylindrical membrane to a drastically narrower catenoid-shaped neck within the spontaneous hemi-fission regime, in a manner that depends critically on the length of the confined tube. These results indicate how the combination of dynamin assembly, and paradoxically disassembly, can lead to diverse pathways to scission. TeaserDynamin mechano-constriction by assembly and curvature-driven instability by free monomers synergistically drive mitochondrial fission.

biophysics↗

bioRxiv2024

Chemokines Kill Bacteria by Binding Anionic Phospholipids without Triggering Antimicrobial Resistance

Classically, chemokines coordinate leukocyte trafficking during immune responses; however, many chemokines have also been reported to possess direct antibacterial activity in vitro. Yet, the bacterial killing mechanism of chemokines and the biochemical properties that define which members of the chemokine superfamily are antimicrobial remain poorly understood. Here we report that the antimicrobial activity of chemokines is defined by their ability to bind phosphatidylglycerol and cardiolipin, two anionic phospholipids commonly found in the bacterial plasma membrane. We show that only chemokines able to bind these two phospholipids kill Escherichia coli and Staphylococcus aureus and that they exert rapid bacteriostatic and bactericidal effects against E. coli with a higher potency than the antimicrobial peptide beta-defensin 3. Furthermore, our data support that bacterial membrane cardiolipin facilitates the antimicrobial action of chemokines. Both biochemical and genetic interference with the chemokine-cardiolipin interaction impaired microbial growth arrest, bacterial killing, and membrane disruption by chemokines. Moreover, unlike conventional antibiotics, E. coli failed to develop resistance when placed under increasing antimicrobial chemokine pressure in vitro. Thus, we have identified cardiolipin and phosphatidylglycerol as novel binding partners for chemokines responsible for chemokine antimicrobial action. Our results provide proof of principle for developing chemokines as novel antibiotics resistant to bacterial antimicrobial resistance mechanisms.

microbiology↗

bioRxiv2024

Kilohertz volumetric imaging of in-vivo dynamics using squeezed light field microscopy

Volumetric functional imaging of transient cellular signaling and motion dynamics poses a significant challenge to current microscopy techniques, primarily due to limitations in hardware bandwidth and the restricted photon budget within short exposure times. In response to this challenge, we present squeezed light field microscopy (SLIM), a computational imaging method that enables rapid detection of high-resolution three-dimensional (3D) light signals using only a single, low-format camera sensor area. SLIM pushes the boundaries of 3D optical microscopy, achieving over one thousand volumes per second across a large field of view of 550 m in diameter and 300 m in depth with a spatial resolution of 3.6 m laterally and 6 m axially. Using SLIM, we demonstrated blood cell velocimetry across the embryonic zebrafish brain and in a free-moving tail exhibiting high-frequency swinging motion. The millisecond temporal resolution also enables accurate voltage imaging of neural membrane potentials in the leech ganglion, and in the hippocampus of behaving mice. These results collectively establish SLIM as a versatile and robust imaging tool for high-speed microscopy applications.

bioengineering↗

bioRxiv2023

How individual P. aeruginosa cells with diverse stator distributions collectively form a heterogeneous macroscopic swarming population

Swarming is a macroscopic phenomenon in which surface bacteria organize into a motile population. The flagellar motor that drives swarming in Pseudomonas aeruginosa is powered by stators MotAB and MotCD. Deletion of the MotCD stator eliminates swarming, whereas deletion of the MotAB stator enhances swarming. Interestingly, we measured a strongly asymmetric stator availability in the WT strain, with MotAB stators produced [~]40-fold more than MotCD stators. However, recruitment of MotCD stators in free swimming cells requires higher liquid viscosities, while MotAB stators are readily recruited at low viscosities. Importantly, we find that cells with MotCD stators are [~]10x more likely to have an active motor compared to cells without, so wild-type, WT, populations are intrinsically heterogeneous and not reducible to MotAB-dominant or MotCD-dominant behavior. The spectrum of motility intermittency can either cooperatively shut down or promote flagellum motility in WT populations. In P. aeruginosa, transition from a static solid-like biofilm to a dynamic liquid-like swarm is not achieved at a single critical value of flagellum torque or stator fraction but is collectively controlled by diverse combinations of flagellum activities and motor intermittencies via dynamic stator recruitment. Experimental and computational results indicate that the initiation or arrest of flagellum-driven swarming motility does not occur from individual fitness or motility performance but rather related to concepts from the jamming transition in active granular matter. ImportanceAfter extensive study, it is now known that there exist multifactorial influences on swarming motility in P. aeruginosa, but it is not clear precisely why stator selection in the flagellum motor is so important or how this process is collectively initiated or arrested. Here, we show that for P. aeruginosa PA14, MotAB stators are produced [~]40-fold more than MotCD stators, but recruitment of MotCD over MotAB stators requires higher liquid viscosities. Moreover, we find the unanticipated result that the two motor configurations have significantly different motor intermittencies, the fraction of flagellum-active cells in a population on average, with MotCD active [~]10x more often than MotAB. What emerges from this complex landscape of stator recruitment and resultant motor output is an intrinsically heterogeneous population of motile cells. We show how consequences of stator recruitment led to swarming motility, and how they potentially relate to surface sensing circuitry.

microbiology↗

bioRxiv2021

Highly basic clusters in the HSV-1 nuclear egress complex drive membrane budding by inducing lipid ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in-vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an "off" switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

microbiology↗

bioRxiv2020

Fine tuning cyclic-di-GMP signaling in Pseudomonas aeruginosa using the type 4 pili alignment complex

To initiate biofilm formation it is critical for bacteria to sense a surface and respond precisely. Type 4 pili (T4P) have been shown to be important in surface sensing, however, mechanism(s) driving downstream changes important for the switch to biofilm growth have not been clearly defined. Here, using macroscopic bulk assays and single cell tracking analyses of Pseudomonas aeruginosa, we uncover a new role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two hybrid and bimolecular fluorescence complementation assays show that PilO physically interacts with SadC and that the PilO-SadC interaction inhibits SadCs activity resulting in decreased biofilm formation and increased motility. We show that disrupting the PilO-SadC interaction contributes to greater variation of cyclic-di-GMP levels among cells, thereby increasing cell-to-cell heterogeneity in the levels of this signal. Thus, this work shows that P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this nucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement. Significance StatementT4P of P. aeruginosa are important for surface sensing and regulating intracellular cyclic-di-GMP levels. This work identifies a new role for the T4P alignment complex, previously known for its role in supporting pili biogenesis, in surface-dependent signaling. Furthermore, our findings indicate that P. aeruginosa uses a single DGC, via a complex web of protein-protein interactions, to integrate signaling through the T4P and the flagellar motor to fine-tune cyclic-di-GMP levels. A key implication of this work is that more than just regulating signal levels, cells must modulate the dynamic range of cyclic-di-GMP to precisely control the transition to a biofilm lifestyle.

microbiology↗

bioRxiv2019

Phenotypic differences in reversible attachment behavior reveal distinct P. aeruginosa surface colonization strategies

Despite possessing the machinery to sense, adhere to, and proliferate on surfaces, it is commonly observed that bacteria initially have a difficult time attaching to a surface. Before forming a bacterial biofilm, planktonic bacteria exhibit a random period of transient surface attachment known as \"reversible attachment\" which is poorly understood. Using community tracking methods at single-cell resolution, we examine how reversible attachment progresses during initial stages of surface sensing. Pseudomonas aeruginosa strains PAO1 and PA14, which exhibit similar exponential trends of surface cell population increase, show unanticipated differences when the behavior of each cell was considered at the full lineage level and interpreted using the unifying quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two complementary surface colonization strategies, which are roughly analogous to \"immediate-\" vs \"deferred-gratification\" in a prototypical cognitive-affective processing system. PAO1 lineages commit relatively quickly to a surface compared to PA14 lineages. PA14 lineages allow detaching cells to retain memory of the surface so that they are primed for improved subsequent surface attachment. In fact, it is possible to identify motility suppression events in PA14 lineages in the process of surface commitment. We hypothesize that these contrasting strategies are rooted in downstream differences between Wsp-based and Pil-Chp-based surface sensing systems.

microbiology↗

bioRxiv2019

Prototypical pacemaker neurons are immunocompetent cells

Pacemaker neurons exert control over neuronal circuit function by their intrinsic ability to generate rhythmic bursts of action potential. Recent work has identified rhythmic gut contractions in human, mice and hydra to be dependent on both neurons and the resident microbiota. However, little is known about the evolutionary origin of these neurons and their interaction with microbes. In this study, we identified and functionally characterized prototypical ANO/SCN/TRPM ion channel expressing pacemaker cells in the basal metazoan Hydra by using a combination of single-cell transcriptomics, immunochemistry, and functional experiments. Unexpectedly, these prototypical pacemaker neurons express a rich set of immune-related genes mediating their interaction with the microbial environment. Functional experiments validated a model of the evolutionary emergence of pacemaker cells as neurons using components of innate immunity to interact with the microbial environment and ion channels to generate rhythmic contractions.

developmental biology↗

bioRxiv2019

Heterogeneity in surface sensing produces a division of labor in Pseudomonas aeruginosa populations

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. The current thinking in the field is that once cells attach to a surface, they uniformly respond with elevated c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

microbiology↗