Biology subjects

Sikirzhytski, V.

Publications and source records attributed to Sikirzhytski, V..

7 recordsLinked to original sources

bioRxiv2026

Spatially Constrained Monte Carlo Permutation Test Reveals Diffusion Changes Near Stress Granules

Intracellular diffusion is inherently heterogeneous, yet single-particle tracking (SPT) analyses are often summarized using cell-wide average parameters that can obscure localized effects. Here, we tracked 40-nm genetically encoded multimeric (GEM) nanoparticles during stress granule (SG) formation and developed SPaCe-MC (Spatially Constrained Monte Carlo permutation test), a statistical framework that generates cytoplasm-specific null models to test whether diffusion associated with a specific cellular structure differs from that expected in the surrounding heterogeneous cytoplasm. Across three SG-inducing conditions, including oxidative stress, DDX3 inhibition, and combined treatment, bulk cytoplasmic analyses revealed distinct responses ranging from increased nanoparticle mobility to increased subdiffusive behavior. In contrast, SPaCe-MC consistently detected a local diffusion constraint in SG-associated regions relative to their treatment-matched cytoplasmic background, revealing a conserved local diffusion effect despite divergent global cytoplasmic responses. Together, our findings establish SPaCe-MC as a framework for identifying compartment-specific diffusion changes in heterogeneous cellular environments.

cell biology↗

bioRxiv2024

Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

Single particle tracking (SPT) is a powerful technique for probing the diverse physical properties of the cytoplasm. Genetically encoded nanoparticles provide an especially convenient tool for such investigations, as they can be expressed and tracked in cells via fluorescence. Among these, 40-nm GEMs provide a unique opportunity to explore the cytoplasm. Their size corresponds to that of ribosomes and big protein complexes, allowing us to investigate the effects of the cytoplasm on the diffusivity of these objects while excluding the influence of chemical interactions during stressful events and pathological conditions. However, it has been shown that cytoplasmic viscosity is tightly regulated and plays a crucial role in maintaining homeostasis during protein synthesis and degradation. Despite this, the effects of GEM expression levels on diffusivity remain largely uncharacterized in mammalian cells. To optimize the GEMs tracking and estimate GEMs-expression effects we constructed dox-inducible GEM expression system and compare with a previously reported constitutive expression system. The optimized level of GEMs expression increases the measured diffusivity from 0.29 {+/-} 0.02 m2/sec in GEMs-overexpressed cells to 0.35 {+/-} 0.02 m2/sec; improve homogeneity throughout the cell population; and facilitates particle tracking. We also improved the analyses of GEM diffusivity by applying effective diffusion coefficient while considering the type of motion and assessing the heterogeneity in the type of motion by calculating the standard deviations of particle displacements. Statement of significanceDescribing cytoplasmic properties, such as environmental viscosity and protein complex motion, is essential for understanding molecular-level changes in cell function and pathology. A recently developed approach uses self-assembling fluorescent protein probes, expressed in cells, to investigate cytoplasmic properties through single-particle tracking (SPT). One such system employs genetically encoded multimeric (GEM) nanoparticles-- scaffold protein structures similar in size to ribosomes. This study addresses a key limitation in SPT of GEMs by examining how varying GEM expression levels affect measured diffusivity and tracking quality in mammalian cells. Our findings demonstrate that controlled GEM expression reduces particle overcrowding, increases measured diffusivity, and enhances track detection. This work contributes valuable insights into optimizing GEM nanoparticle applications for studying cytoplasmic viscosity and motion dynamics.

biophysics↗

bioRxiv2023

Suppression of HIV and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection, marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for effective therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of the HIV and cocaine-induced transcriptomes in primary cortical cultures revealed a significant overexpression of the macrophage-specific gene, aconitate decarboxylase 1 (Acod1), caused by the combined insults of HIV and cocaine. ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. The itaconate produced facilitates cytokine production and subsequently activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. While the role of itaconate in limiting inflammation has been studied in peripheral macrophages, its immunometabolic function remains unexplored in HIV and cocaine-exposed microglia. We assessed in this model system the potential of 4-octyl-itaconate (4OI), a cell-penetrable esterified form of itaconate known for its potent anti-inflammatory properties and potential therapeutic applications. We administered 4OI to primary cortical cultures exposed to Tat and cocaine. 4OI treatment increased the number of microglial cells in both untreated and Tat{+/-}Cocaine-treated cultures and also reversed the morphological altercations induced by Tat and cocaine. In the presence of 4OI, microglial cells also appeared more ramified, resembling the quiescent microglia. Consistent with these results, 4OI treatment inhibited the secretion of the proinflammatory cytokines IL-1, IL-1{beta}, IL-6, and MIP1- induced by Tat and cocaine. Transcriptome profiling further determined that Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (Nqo1), Glutathione S-transferase Pi (Gstp1), and glutamate cysteine ligase catalytic (Gclc), were most significantly activated in Tat-4OI treated cultures, relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development aimed at addressing HAND coupled with CUD comorbidities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/559154v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bfffa8org.highwire.dtl.DTLVardef@129ad8dorg.highwire.dtl.DTLVardef@1412ea2org.highwire.dtl.DTLVardef@5cb968_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Model of 4OI-mediated neuroprotection against Tat-Cocaine toxicity Tat and Tat-Cocaine treatment induce neuronal damage, which is mitigated by 4OI through microglia cells. This cartoon shows the reduction of harmful effects such as proinflammatory cytokine release, upregulation of P2R, PDE, and Acod1 by the presence of 4OI. This ester modified itaconate triggers anti-inflammatory responses and activates antioxidant pathways. C_FIG

neuroscience↗

bioRxiv2023

CDK19 and CDK8 Mediator kinases drive androgen-independent in vivo growth of castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) remains incurable due to its high plasticity. We found that Mediator kinases CDK8 and CDK19, pleiotropic regulators of transcriptional reprogramming, are differentially affected by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, expression of CDK8 decreases while CDK19 increases during prostate carcinogenesis, but both CDK19 and CDK8 are upregulated in metastatic CRPC. Genetic inactivation of CDK8 and CDK19 suppresses CRPC tumor growth in castrated male mice and renders CRPC responsive to androgen deprivation. Restoration of active CDK19 or CDK8 kinases reverses this phenotype, indicating that CRPC becomes dependent on Mediator kinase activity for in vivo growth under the conditions of androgen deprivation. Selective CDK8/19 inhibitors suppress androgen-independent growth of cell line-based and patient-derived CRPC xenografts, whereas prolonged inhibitor treatment induces tumor regression and even leads to cures. Mediator kinase activity was found to affect tumor and stromal gene expression preferentially in castrated mice, orchestrating castration-induced transcriptional reprogramming. These results warrant the exploration of Mediator kinase inhibitors for CRPC therapy.

cancer biology↗

bioRxiv2022

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification

Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. However, there are no effective strategies targeting cancer cells with CA while sparing normal cells. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) to be overexpressed in tumors with CA, and its high expression is associated with dramatically worse clinical outcome. We demonstrated that TACC3 forms distinct functional interactions in mitotic and non-mitotic cancer cells with CA to facilitate centrosome clustering (CC) and transcriptional repression of tumor suppressors, respectively. We showed, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA and inhibition of TACC3 blocks this interaction, leading to apoptosis via multipolar spindle formation and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis. In interphase, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in nucleus, and its inhibition causes p53-independent G1 arrest and apoptosis by blocking these interactions and activating the transcription of key tumor suppressors (e.g., p21, p16 and APAF1). Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition. Targeting TACC3 by small molecule inhibitors or guide RNAs strongly inhibits growth of organoids and breast cancer cell line- and patient-derived xenografts with CA. Altogether our results pave the way towards therapeutic targeting of TACC3 in highly aggressive cancers.

cancer biology↗

bioRxiv2019

Inhibition of the DDX3 prevents HIV-1 Tat and cocaine-induced neurotoxicity by targeting microglia activation

HIV-1 Associated Neurocognitive Disorder (HAND) is commonly seen in HIV-infected patients. Viral proteins including Tat cause neuronal toxicity and is worsened by drugs of abuse. To uncover potential targets for anti-HAND therapy, we employed a literature mining system, MOLIERE. Here, we validated Dead Box RNA Helicase 3 (DDX3) as a target to treat HAND via a selective DDX3 inhibitor, RK-33. The combined neurotoxicity of Tat protein and cocaine was blocked by RK-33 in rat and mouse cortical cultures. Transcriptome analysis showed that Tat-activated transcripts include makers and regulators of microglial activation, and RK-33 blocked Tat-induced activation of these mRNAs. Elevated production of proinflammatory cytokines was also inhibited by RK-33. These findings show that DDX3 contributes to microglial activation triggered by Tat and cocaine, and DDX3 inhibition shows promise as a therapy for HAND. Moreover, DDX3 may contribute to the pathology of other neurodegenerative diseases with pathological activation of microglia.

neuroscience↗

bioRxiv2018

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls mitosis through assembly of a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1, 2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4-9]. Through gene editing and live-cell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons. We also show that RZZ forms the mesh-like fibrous corona, a structural expansion of the outer kinetochore important for timely chromosome congression [10-13] once Mps1 phosphorylates the N-terminus of Rod. Artificially tethering Mad1-Mad2 to kinetochores enabled long-term mitotic arrest in the absence of RZZ. Conversely, blocking early RZZ-independent recruitment of Mad1-Mad2 eliminated the transient SAC response in RZZ-null cells. We conclude that RZZ drives structural changes in the outer kinetochore that facilitate chromosome bi-orientation and chronic SAC transduction, a key determinant of cytotoxicity during anti-mitotic drug therapy [14-16].

cell biology↗