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Ho, C.-M.

Publications and source records attributed to Ho, C.-M..

11 recordsLinked to original sources
bioRxiv2026

Characterization of Host Cell Cytosol-filled Vesicles in the Human Malaria Parasite Plasmodium falciparum

Endocytosis of host cell cytosol is a key process in malaria blood stages. The endocytosed material consists mostly of haemoglobin (Hb) and is transported to the parasite's digestive vacuole (DV), where it is degraded. However, the endosomal transport pathway of the parasite is not well defined. A number of proteins are known that - when inactivated - lead to the appearance of Hb-filled vesicles (HbVs) in the parasite cytoplasm and prevent Hb from arriving in the DV, indicating they play a role in endosomal transport. However, despite the prominence of these HbVs, their morphology, molecular composition, and relationship to the endosomal transport pathway remain poorly understood. Here we carried out a morphological and surface proteome study of HbVs. CryoET showed HbVs are coatless, double membraned vesicles with a very narrow inter-membrane space devoid of larger protein densities. HbV surface proteomes generated by BioIDs most prominently detected DV proteases. Halo-based tracking of one of these DV protease confirmed it as a bona fide HbV cargo. The BioID also identified a number of vesicle trafficking proteins, including PfTBC8 and Rab11 proteins. Functional analysis of PfTBC8 showed its importance for the transport of Hb to the DV. This was due to a novel phenotype characterized by accumulation of diverse Hb-filled structures in the parasite cytosol and gradual decline of DV protease trafficking. PfTBC8 DiQ-BioID detected Rab11a and PfTBC8 inactivation altered Rab11a localization, suggesting it may be a Rab11a effector and that the new endosomal transport phenotype may result from a recycling defect. Together with the detection of VPS35 in the BioID and evidence from the CryoET of a disrupted inner HbV membrane in a proportion of vesicles, these findings support a model in which HbVs undergo maturation prior to fusion with the DV. Overall, our findings define morphological and molecular features of HbVs, identify protein cargo transported through this pathway, and reveal a previously unrecognized Rab-regulatory component required for endosomal trafficking in blood-stage P. falciparum parasites.

microbiology↗
bioRxiv2026

Imaging large fields-of-view at high resolution in cryo-ET with square beam montaging

Visualizing macromolecules within their native cellular context by cryo-electron tomography (cryo-ET) is fundamentally limited by the trade-off between field of view and resolution: Capturing high-resolution information about biomolecules requires high magnification, which restricts the field of view and obscures the cellular context in which those biomolecules function. Collecting montage data by tiling the electron beam over the region of interest offers one solution, although traditional round electron beams cause excessive radiation damage across overlapping regions. We previously made electron beams square in shape, enabling montage collection with minimal overlap and thereby reducing excessive exposure and loss of high-resolution information. Here, we create a pipeline for collecting and processing montage cryo-ET data with square electron beams. We show that square beam montages retain high-resolution information by reconstructing virus-like particles to 3.5 [A] resolution using sub-tomogram averaging, and apply the workflow to imaging a glial cell and malaria parasite lamellae over fields of view up to 65 m2. We also provide a comprehensive protocol to make square beams accessible to the community.

molecular biology↗
bioRxiv2026

Long-term memory performance optimization via Neural network-based curve fitting in Drosophila

Long-term memory (LTM) formation typically requires extensive training or highly salient experiences, limiting learning efficiency. Operant conditioning is generally thought to produce stronger memory than classical conditioning because of its active learning component. Unexpectedly, however, laser-based social conditioning in Drosophila melanogaster revealed that while classical paradigms yielded lower short-term memory (STM) scores but higher LTM retention, operant paradigms exhibited higher STM scores followed by rapid LTM decay. To resolve this discrepancy, we employed the AI Complex Systems Response (AI-CSR) framework, which reconstructs high-dimensional learning landscapes from sparse sampling and predicts globally optimal training conditions. Following AI-CSR optimization, operant conditioning produced a twofold increase in LTM scores, yielding the strongest 24-hour social memory performance reported in flies to date and revealing the expected superiority of active learning, which had conversely shown poorer performance under standard training protocols. In contrast, AI-CSR did not further enhance classical conditioning performance but reduced training time by 50%. Single-cell RNA sequencing revealed expanded neuronal recruitment marked by the activation and inhibition of various gene combinations. Together, these findings link circuit-level reorganization with molecular programs underlying efficient long-term memory and demonstrate how AI-guided optimization can uncover latent learning capacity in biological systems.

animal behavior and cognition↗
bioRxiv2025

Endogenous structure of antimalarial target PfATP4 reveals new class of apicomplexan P-type ATPase modulators

The Plasmodium falciparum sodium efflux pump PfATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7[A] cryoEM structure of PfATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, PfABP, which forms a conserved, likely modulatory interaction with PfATP4. The discovery of PfABP presents a new avenue for designing novel PfATP4 inhibitors.

microbiology↗
bioRxiv2023

In situ cryoET reveals inhibitor-induced perturbations in Pf80S GTPase interactions

Malaria parasites rely heavily on rapid, high fidelity protein synthesis to infect and replicate in human erythrocytes, making translation an attractive target for new antimalarials. Here, we have determined in situ structures of Pf80S ribosomes in thirteen conformational and compositional states from cryoFIB-milled Plasmodium falciparum-infected human erythrocytes across the stages of asexual intraerythrocytic parasite replication. We observe eight active translation intermediates, enabling us to define the native malarial translation elongation cycle, which surprisingly features a bifurcation at the decoding stage of the cycle that has not previously been described. Examination of perturbations in the distribution of ribosomes among these states in the presence of a malaria-specific translation inhibitor suggests that the inhibitor impedes PfeEF2 and PfeEF1 interactions with the ribosome. We integrated our in situ cryoET data with proteomic and ultrastructural data to arrive at a deeper understanding of malarial translation, which will inform development of new therapies.

biophysics↗
bioRxiv2022

Central visceral commands formatted as the slow rhythms encoded in the temporal structures of the sympathetic firing originated from the neonatal rat spinal cord in vitro

Central sympathetic neural circuits continuously generate efferent commands to sustain rhythmic operations of their peripheral effectors. However, beyond the firing rates, in what configuration an effective visceral command is formatted by the spiking activities was largely unknown. This study used an in vitro splanchnic nerve-spinal cord preparation of the neonatal rats as an experimental model and recorded spontaneous efferent activities from single sympathetic fibers. The patterns of the fiber activities were quantitatively evaluated by a metric, the so-called local variation (Lv). Lv was derived from calculating the relative differences between adjacent interspike intervals (ISIs), and thus, described the spiking patterns from a regular spiking with Lv = 0 to that spiking in bursts with Lv >1. Along the time course, the dynamic components of Lv (dLv) displayed quasiperiodic oscillations. Continuous wavelet analysis showed that dLv oscillations registered a dominant power rhythm at [~]7 mHz. This slow rhythmicity was heterogeneously altered by application of various antagonists that interrupted endogenous neurotransmitter activities mediated by ionotropic glutamate receptors or GABAA receptors in the spinal cord. Thus, the oscillation of dLv manifested itself as a feature of neural network operation. On the assumption that the total power of the dLv oscillations reflects an activity status of neural network operation, the antagonist-induced change of the power fits well with a concomitant ISI change in a negative sigmoid relationship, which explains the heterogeneity of the antagonist-induced firing responses. In conclusion, dLv oscillations reflect a dynamic status of neural network operation. The slow rhythms embedded in the dLv oscillations are likely acting as an information coder and convey effective central visceral commands that can be followed by their downstream effectors. Author summaryIn what format the information is encoded by the neural activity is relatively unknown. This is especially true for autonomic regulation of visceral functions. We seek to determine the format of the central sympathetic commands, by which it can generate an effective driving force to regulate the operation of their peripheral target organs. Because most visceral organs operate with certain rhythms, we anticipate that the central visceral commands are also formatted in rhythms. Two aspects of techniques were employed. One was the in vitro electrophysiological and pharmacological techniques to acquire and to manipulate the neural signals recorded from sympathetic single-fibers. The other was the computational techniques to extract the information embedded in the timing of the spiking behaviors and to examine how these features were altered by pharmacological manipulations. We found a spontaneous change of sympathetic spiking patterns displaying rhythms with a frequency that could match the rhythmic operation of many visceral organs. Thus, the meaningful information carried by the sympathetic neural circuits for the visceral controls is likely to be encoded in the dynamics of their spiking patterns. In summary, a dynamic change of neural spiking patterns could be used as a simple scheme for neural information coding.

neuroscience↗
bioRxiv2021

Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition

The RhopH complex is implicated in malaria parasites ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1 and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo electron microscopy, mass spectrometry, and the cryoID program. This first direct observation of an exported P. falciparum transmembrane protein--in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices--offers insights into assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.

biophysics↗
bioRxiv2019

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multidrug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA and compound 2 is an uncompetitive inhibitor targeting the LpxA-product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1, and was optimized using structural information to achieve improved antibacterial activity against wild type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme-product complexes in drug discovery.

biochemistry↗
bioRxiv2019

Subcellular localization of drug distribution by super-resolution ion beam imaging

Technologies that visualize multiple biomolecules at the nanometer scale in cells will enable deeper understanding of biological processes that proceed at the molecular scale. Current fluorescence-based methods for microscopy are constrained by a combination of spatial resolution limitations, limited parameters per experiment, and detector systems for the wide variety of biomolecules found in cells. We present here super-resolution ion beam imaging (srIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added small molecules. Uniquely, the atomic constituents of the biomolecules themselves can often be used in our system as the "tag". We visualized the subcellular localization of the chemotherapy drug cisplatin simultaneously with localization of five other nuclear structures, with further carbon elemental mapping and secondary electron visualization, down to [~]30 nm lateral resolution. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. These data highlight how multiplexed super-resolution techniques, such as srIBI, will enable studies of biomolecule distributions in biologically relevant subcellular microenvironments. One Sentence SummaryThree-dimensional multiplexed mass spectrometry-based imaging revealed the subcellular localization of proteins and small molecules at super-resolution.

systems biology↗
bioRxiv2018

Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-m resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.

bioengineering↗