Biology subjects

Sha, H.

Publications and source records attributed to Sha, H..

10 recordsLinked to original sources

bioRxiv2026

Multi-channel high-density single-molecule localization

Deep learning has enabled single-molecule localization microscopy (SMLM) at high emitter densities, but only for single channel systems. Here we present DECODE-Plex, a deep-learning-based framework to localize dense single molecules with overlapping point spread functions simultaneously in multiple channels. We showcase DECODE-Plex on experimental ultra-high density dual-color and 3D live-cell data. Packaged for ease of use, it will enable many groups to improve imaging speed and quality of multi-channel SMLM.

biophysics↗

bioRxiv2026

Experimental Context Shapes PRR-Mediated Immune Output Sensitivity in Arabidopsis

Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeqcyt/pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

plant biology↗

bioRxiv2024

Single molecule spectrum dynamics imaging with 3D target-locking tracking

Fluorescence spectrum can provide rich physicochemical insights into molecular environments and interactions. However, imaging the dynamic fluorescence spectrum of rapidly moving biomolecules, along with their positional dynamics, remains a significant challenge. Here, we report a three-dimensional (3D) target-locking tracking-based single molecule fluorescence Spectrum Dynamics Imaging Microscopy (3D-SpecDIM), which is capable of simultaneously capturing both the rapid 3D positional dynamics and the physicochemical parameters changing dynamics of the biomolecules with enhanced spectral accuracy, high spectral acquisition speed, single-molecule sensitivity, and high 3D spatiotemporal localization precision. As a demonstration, 3D-SpecDIM is applied to real-time spectral imaging of the mitophagy process, showing its enhanced ratiometric fluorescence imaging capability. Additionally, 3D-SpecDIM is used to perform multi-resolution imaging, which provides valuable contextual information on the mitophagy process. Furthermore, we demonstrated the quantitative imaging capability of 3D-SpecDIM by imaging the cellular blebbing process. By continuously monitoring the physicochemical parameter dynamics of biomolecular environments through spectral information, coupled with 3D positional dynamics imaging, 3D-SpecDIM offers a versatile platform for concurrently acquiring multiparameter dynamics, providing comprehensive insights unattainable through conventional imaging techniques. 3D-SpecDIM represents a substantial advancement in single-molecule spectral dynamics imaging techniques.

biophysics↗

bioRxiv2024

Unlocking expanded flagellin perception through rational receptor engineering

The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologs possess narrow perception ranges, but four with expanded perception ranges have been identified. Using diversity analyses, AlphaFold-Multimer modeling, and amino acid properties, key residues enabling expanded recognition were mapped to FLS2s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) and FLS2XL (Vitis riparia) into homologs with canonical perception. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22s C-terminus, suggesting more alleles with expanded perception exist. Our experimental data enabled the identification of specific receptor amino acid properties and AlphaFold3 metrics that facilitate predicting FLS2-flg22 recognition. This study provides a framework for rational receptor engineering to enhance pathogen restriction.

plant biology↗

bioRxiv2023

AutoUnmix: an autoencoder-based spectral unmixing method for multi-color fluorescence microscopy imaging

Multiplexed fluorescence microscopy imaging is widely used in biomedical applications. However, simultaneous imaging of multiple fluorophores can result in spectral leaks and overlapping, which greatly degrades image quality and subsequent analysis. Existing popular spectral unmixing methods are mainly based on computational intensive linear models and the performance is heavily dependent on the reference spectra, which may greatly preclude its further applications. In this paper, we propose a deep learning-based blindly spectral unmixing method, termed AutoUnmix, to imitate the physical spectral mixing process. A tranfer learning framework is further devised to allow our AutoUnmix adapting to a variety of imaging systems without retraining the network. Our proposed method has demonstrated real-time unmixing capabilities, surpassing existing methods by up to 100-fold in terms of unmixing speed. We further validate the reconstruction performance on both synthetic datasets and biological samples. The unmixing results of AutoUnmix achieve a highest SSIM of 0.99 in both three- and four-color imaging, with nearly up to 20% higher than other popular unmixing methods. Due to the desirable property of data independency and superior blind unmixing performance, we believe AutoUnmix is a powerful tool to study the interaction process of different organelles labeled by multiple fluorophores.

cell biology↗

bioRxiv2023

Deep learning-enhanced single-molecule spectrum imaging

Fluorescence is widely used in biological imaging and biosensing. Rich information can be revealed from the fluorescence spectrum of fluorescent molecules, such as pH, viscosity and polarity of the molecules environment, and distance between two FRET molecules. However, constructing the fluorescence spectrum of a single fluorescent molecule typically requires a significant number of photons, which can suffer from photobleaching and therefore limit its potential applications. Here we propose a deep learning-enhanced single-molecule spectrum imaging method (SpecGAN) for improving the single-molecule spectrum imaging efficiency. In SpecGAN, the photon flux required to extract a single-molecule fluorescence spectrum can be reduced by 100 times, which enables it two orders of magnitude higher temporal resolution compared to the conventional single-molecule spectrometer. The concept of SpecGAN was validated through numerical simulation and single Nile Red molecule spectrum imaging on support lipid bilayers (SLBs). With SpecGAN, the super-resolution spectrum image of the COS-7 membrane can be reconstructed with merely 12,000 frames of single-molecule localization images, which is almost half of the previously reported frame count for spectrally resolved super-resolution imaging. The low photon flux requirement and high temporal resolution of SpecGAN make it a promising tool for investigating the molecular spectrum dynamics related to biological functions or biomolecule interactions.

biophysics↗

bioRxiv2022

Germline-soma Supply Mitochondria for mtDNA Inheritance in Mouse Oogenesis

Maternal transmission paradigm of mtDNA remains controversial in mammalian oogenesis. Germline-soma-to-oocyte communication by numerous transzonal nanotubes (TZTs) reminds whether intercellular mitochondrial transfer is associated with maternal inheritance. Here, we found that mouse oocytes egocentrically receive mitochondria via TZTs, which projected from germline-soma, to achieve 105 copies, instead of de novo synthesis of mtDNA subpopulation in growing oocytes. De novo assembled TZTs amongst germline-soma and oocytes accumulated mtDNA amounts of the oocytes in vitro. However, mitochondrial supplement from germline-soma gradually diminished along with oocyte growth and was terminated by meiosis resumption, in line with a decrease in the proportion of germline-soma with thriving mtDNA replication and FSH capture capability. Thus, germline-soma-to-oocyte mitochondrial transfer is responsible for mammalian mtDNA inheritance as well as oogenesis and aging. One-sentence summariesThe maternal mtDNA transmission accompanied by oogenesis is inseparable from germline-soma mitochondrial transport.

developmental biology↗

bioRxiv2020

Germline Selection by Meiosis Defends the Transmission of defective Mitochondria with mtDNA variants

Germline selection of mtDNA is vital in maternal inheritance of mtDNA, as it can eliminate severe mtDNA mutations. However, current evidence concerning germline selection at meiosis level comes from incomplete mtDNA sequencing in human first polar body (PB1), which lacks persuasion. Here, we found various variants, including pathogenic mutation sites, present on whole genome of mtDNA in human PB1 compared with its oocyte. And that PB1 mitochondria with mtDNA variants were defective. Afterwards, to further explore how mitochondria enter PB, the defective mitochondria transfer in mouse germline, including cumulus-oocyte-complexes at germinal vesicle and matured oocytes stage. It confirmed that in the first and second meiosis, active purification selected defective mitochondria into PB1 and PB2. Thus, twice meiosis is the last defense system for purifying selection of mtDNA mutations during oogenesis, which also demonstrated that PB1 and PB2 would be final destination of deleterious mtDNA mutations in germline selection.

cell biology↗

bioRxiv2019

Suppression of Docetaxel induced apoptosis in Survivin-depleted cells due to failure of sustained mitotic block

The antitumor effect of taxanes have been attributed to their ability to induce mitotic arrest through activation of the spindle assembly checkpoint. Cell death following prolonged mitotic arrest is mediated by the intrinsic apoptosis pathway. Accordingly, factors that influence the robustness of mitotic arrest or disrupt the apoptotic machinery might confer drug resistance. Survivin is an inhibitor of apoptosis protein. Its overexpression has been associated with resistance to multiple anticancer agents including taxanes, and its targeting led to drug sensitization. On the other hand, Survivin is a key regulator of mitosis, which is shown to be required for stable activation of the spindle assembly checkpoint. Since the sensitivity of taxanes depends on a functional spindle checkpoint, inhibition of Survivin may lead to drug resistance, which is the opposite effect of its anti-apoptotic function. Here we show that Survivin-depleted cells escape the mitotic block following Docetaxel treatment, thereby evading drug induced apoptosis. Moreover, Survivin depletion increases the level of mitotic catastrophe and cellular senescence induced by Docetaxel and enhanced its efficacy against clonogenic survival of tumor cells. Our finding suggests that inhibition of Survivin promotes non-apoptotic mechanisms following Docetaxel treatment rather than increases the sensitivity of apoptosis.

cancer biology↗

bioRxiv2018

Inhibitor of the spindle assembly checkpoint surpasses apoptosis sensitizer in synergy with taxanes

The antitumor effect of taxanes have been attributed to their ability to induce mitotic arrest through activation of the spindle assembly checkpoint. Cell death following prolonged mitotic arrest is mediated by the intrinsic apoptosis pathway. Thus, apoptosis sensitizers which inhibit antiapoptotic Bcl-2 family proteins has been shown to enhance taxanes-induced cell death. By contrast, spindle checkpoint disruption facilitates mitotic slippage and is thought to promote taxanes resistance. Notably, other modes of cell death also contribute to treatment outcomes. Here we show that inhibition of the spindle checkpoint suppresses taxanes induced apoptosis but increases terminal growth arrest of tumor cells with features of cellular senescence. By using clonogenic assay which measures the net result of multiple forms of cell death and is more reflective of therapeutic response, our finding suggests apoptosis is not a major determinant of antitumor efficacy of taxanes, whereas spindle checkpoint inhibitor displays a long-term advantage over apoptosis sensitizer in blocking colony outgrowth of tumor cells when combined with different microtubule toxins, therefore represents a superior therapeutic strategy.\n\nSIGNIFICANCEApoptosis has long been regarded as the primary mechanism of anti-cancer efficacy of taxanes, while the role of the spindle assembly checkpoint (SAC) in treatment response to taxanes has been controversial. Either apoptosis sensitizer or inhibitor of SAC has been reported to synergize with taxanes. While inhibitor of antiapoptotic proteins potentiates taxanes induced apoptosis, inhibitor of SAC suppresses apoptosis by facilitating mitotic slippage, that is why it is implicated in taxanes resistance. By demonstrating that apoptotic rates are not associate with long-term treatment response, not only do we find that inhibitor of SAC displays a long-term advantage over apoptosis sensitizer in combination with taxanes, but we also resolve the dispute around the role of SAC in cellular response to taxanes.

cancer biology↗