Biology subjects

Mak, M.

Publications and source records attributed to Mak, M..

14 recordsLinked to original sources

bioRxiv2026

Internalized Components of Membrane Attack Complexes Disrupt Proteostasis and Acquire Alarmin-Like Properties

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

biochemistry↗

bioRxiv2026

An optopharmacological tool for on-demand perturbation of branched actin polymerization

Regulated actin cytoskeletal organization and dynamics are essential for life-sustaining processes in eukaryotic cells. The spatiotemporal regulation of filamentous actin can generate a staggering variety of higher-order architectures to meet diverse cellular needs. However, connecting a specific actin substructure to the selective cellular function remains challenging because tools to specifically inhibit F-actin in a spatiotemporally controlled manner are currently limited. To address this crucial technical gap, we have synthesized and characterized a photocrosslinkable small molecule inhibitor of a major and evolutionarily conserved actin nucleation factor, Arp2/3 complex. The new inhibitor "AZ" combines the specificity of a preexisting inhibitor CK666 with photosensitive crosslinking by an Azido group, such that a light-induced, instantaneous, on-demand, irreversible inhibition of Arp2/3 complex-dependent actin nucleation is achieved in a spatiotemporally defined manner at a subcellular, supracellular, and organismal scale. Whole cell treatment with AZ, followed by subcellular photoirradiation, revealed a divergent actin nucleation homeostasis in the cortex of different cells. Thus, AZ could be a powerful tool to infer actin polymerization homeostasis and network properties in live cells-processes which otherwise remain obscure.

cell biology↗

bioRxiv2026

Volumetric mechanoplasticity couples melanoma drug tolerance to susceptibility to CD8+ T cell killing

Cell swelling has been reported in physiological tumor contexts, but whether sustained cell volume expansion leaves a durable state that reshapes drug and immune responses in melanoma is unknown. Using controlled hypotonic dilution as an experimental handle, we compare two priming regimens with comparable hypotonic exposure but different magnitude and persistence of volume expansion, enabling us to test which post-recovery phenotypes track with sustained enlargement beyond hypotonic exposure alone. Sustained enlargement produces a measurable size imprint after return to isotonic conditions, accompanied by nuclear lamina and chromatin remodeling with p53 pathway engagement and suppression of replication and growth programs. After recovery, primed cells reinforce F-actin organization, migrate faster in 3D collagen, and induce antioxidant and anti-ferroptosis defenses consistent with improved stress survival. A gene expression signature derived from this response is associated with poorer outcome in TCGA skin cutaneous melanoma. Unexpectedly, the same volume history also increases IFN-{gamma} response, elevates MHC-I, reduces sialylation, and increases susceptibility to CD8+ T cell killing. These findings indicate that persistent volume history can couple drug tolerance programs to an exploitable increase in immune visibility.

biophysics↗

bioRxiv2025

Loss of cilia drives centriole clustering and elimination during mammalian spermatogenesis

Cilia are microtubule-based organelles essential for signaling and motility, and their dysfunction causes ciliopathies often associated with infertility. In male germ cells, two types of cilia are present: zygotene primary cilia and sperm flagella. To define the role of cilia in spermatogenesis, we conditionally ablated the distal appendage protein CEP164, required for basal body docking and ciliogenesis, in male germ cells. CEP164 localized to the mother centriole/basal body throughout spermatogenesis, and its loss led to male infertility accompanied by absence of both zygotene cilia and sperm flagella. Despite defective ciliogenesis, meiotic chromosome pairing and DNA double-strand break repair proceeded normally. However, round spermatids exhibited basal body docking and flagellogenesis defects, and frequently formed supernumerary centriole clusters that were subsequently eliminated via residual bodies. Live-cell imaging revealed that centrioles were highly mobile, and centriole pairs from neighboring cells often associated through intercellular bridges, forming aggregates. These results establish that basal body docking is crucial for retaining centrioles within spermatids, and its disruption leads to centriole clustering and loss. In contrast, zygotene cilia are dispensable for meiotic chromosome pairing and DNA repair during mammalian spermatogenesis.

developmental biology↗

bioRxiv2025

Development of Collagenous Filaments with Tuneable Mechanical Properties Using a 3D Bioprinter and Molecular Crowding

Engineering collagen constructs that replicate the mechanical strength and biological functionality of load-bearing tissues like tendon remains a significant challenge. This study establishes a scalable and accessible biofabrication method that combines the precision of a commercial 3D bioprinter with the physicochemical principles of molecular crowding. Using an acidic collagen solution in combination with a hypertonic polyethylene glycol (PEG) bath, we systematically investigate how modulating pulling speed and nozzle diameter dictates the final filament properties. Our results demonstrate that faster pulling speeds and smaller nozzle diameters produce thinner, more densely packed filaments. High-resolution scanning electron microscopy (SEM) and polarised light microscopy reveal a tuneable transition in surface morphology, from a disordered isotropic state to a highly ordered phase, quantitatively confirmed by a sharp increase in birefringence. Critically, we demonstrate the preservation of collagens native, periodic 66 nm d-banding, an essential motif for cell interaction. This structural refinement translates to a dramatic enhancement of mechanical performance, with the strongest filaments achieving an ultimate tensile strength of 0.64 GPa and a Youngs modulus of 8.9 GPa, values that surpass native tendon. Furthermore, the fabricated filaments are highly biocompatible and bio-instructive, promoting robust alignment and elongation of mesenchymal stem cells. This work provides a direct link between bioprocess parameters and the resulting hierarchical structure, offering a versatile platform for fabricating high performance, biomimetic materials for regenerative medicine.

bioengineering↗

bioRxiv2025

LIPTER, a cardiomyocyte-enriched long noncoding RNA, controls cardiac cytoskeletal maturation and is regulated by a cardiomyocyte-specific enhancer.

Cardiac development is characterized by a complex series of molecular, cytoskeletal and electrophysiological changes that guarantee the proper functioning of adult cardiomyocytes (CMs). These changes are defined by cell-type-specific transcriptional rewiring of progenitor cells to form CMs, and are regulated by various epigenetic elements, such as long noncoding RNAs (lncRNAs). LncRNAs are versatile epigenetic regulators as they may act in cis or in trans to orchestrate important gene programs during cardiac development and may concurrently encode micropeptides. LIPTER is one such lncRNA, previously shown to regulate lipid droplet transport in cardiomyocytes and thus an important regulator of cardiomyocyte metabolism. Here we show that LIPTER also plays a role in the cytoskeletal maturation of CMs, as loss of LIPTER leads to persistent expression of fetal genes, changes in chromatin accessibility, disorganized sarcomeres and impaired calcium homeostasis in CMs. Furthermore, we have identified a cardiomyocyte-specific regulatory enhancer that regulates the expression of LIPTER in CMs. CRISPR-mediated inhibition of this enhancer led to reduced LIPTER expression in CMs and increased expression of fetal genes. This CM-specific enhancer could therefore be manipulated to control the expression of LIPTER for therapeutic benefit. In summary, we have unravelled a novel role of LIPTER in CMs cytoskeletal maturation and have identified a CM-specific enhancer for LIPTER.

genomics↗

bioRxiv2025

Epidermal Stem Cells Control Periderm Injury Repair via Matrix-Driven Specialization of Intercellular Junctions

Epidermal stem cells interact with the extracellular matrix (ECM) to regulate their differentiation and maintain skin architecture. Here, we demonstrate a novel role for basal epidermal stem cells (BECs)-ECM interaction in regulating adhesion molecules expressed by the periderm--the superficial epidermal cells (SECs) of the embryonic bilayered skin. Using the developing zebrafish fin fold, we identify BECs form distinct regions of collagen-versus laminin-enriched basement membranes through integrin-mediated adhesions. Mechanistically, collagen-associated BECs form desmosomes and adherens junctions (AJs) with SECs while laminin-associated BECs display reduced desmosomes but sustain AJs and actomyosin expression with SECs. Notably, we show both in vivo and in a bilayered human keratinocyte model, that laminin, compared to collagen, is sufficient to repress desmosome formation while sustaining AJs specifically at the interlayer cell contacts. In vivo, laminin deficiency enhances desmosome expression across layers and impairs the wound-healing capacity of SECs. This defect was partially rescued by genetic reduction of the desmosome protein Desmoplakin-1a, highlighting the role of ECM-dependent junctional specialization in mediating differences in SEC injury response. Overall, our findings identify that stem cells, through their matrix, establish specialized junctions in the overlying stratified epithelium, which contribute to skin healing properties.

developmental biology↗

bioRxiv2025

Intracellular Delivery of Bone Nanoparticles to Mitigate Irradiation-Induced Damage in Bone Marrow Mesenchymal Stem Cells

Ionizing radiation (IR)-induced bone damage presents a major clinical challenge by impairing bone marrow function and disrupting normal bone remodeling. Bone regeneration depends on bone marrow-derived mesenchymal stem cells (BMSCs), which are highly sensitive to IR that causes DNA damage, oxidative stress, apoptosis, and a shift from osteogenesis to adipogenesis, ultimately leading to bone loss and impaired healing. This study evaluated the therapeutic potential of intracellularly delivered bone-derived nanoparticles (BPs) in mitigating IR-induced BMSCs damage. We found that IR exposure caused significant BMSCs dysfunction, including reduced proliferation, increased apoptosis, persistent DNA damage, and a shift toward adipogenic differentiation. Treatment with BPs led to efficient intracellular uptake, improved cell morphology, enhanced proliferation, reduced apoptosis, and preservation of balanced differentiation capacity. Transcriptomic analysis via RNA sequencing revealed that BPs restored key molecular pathways disrupted by IR, particularly those involved in cell cycle regulation, extracellular matrix (ECM) remodeling, and apoptosis. By reversing these transcriptional impairments, BPs supported genomic stability and the regenerat ive function of BMSCs. Overall, these findings suggest that BPs effectively counteract IR-induced cellular damage and enhance the regenerative capacity of BMSCs, offering a promising therapeutic strategy for radiation -induced skeletal injuries.

bioengineering↗

bioRxiv2023

Volumetric Compression Shifts Rho GTPase Balance and Induces Mechanobiological Cell State Transition

During development and disease progression, cells are subject to osmotic and mechanical stresses that modulate cell volume, which fundamentally influences cell homeostasis and has been linked to a variety of cellular functions. It is not well understood how the mechanobiological state of cells is programmed by the interplay of intracellular organization and complex extracellular mechanics when stimulated by cell volume modulation. Here, by controlling cell volume via osmotic pressure, we evaluate physical phenotypes (including cell shape, morphodynamics, traction force, and extracellular matrix (ECM) remodeling) and molecular signaling (YAP), and we uncover fundamental transitions in active biophysical states. We demonstrate that volumetric compression shifts the ratiometric balance of Rho GTPase activities, thereby altering mechanosensing and cytoskeletal organization in a reversible manner. Specifically, volumetric compression controls cell spreading, adhesion formation, and YAP nuclear translocation, while maintaining cell contractile activity. Furthermore, we show that on physiologically relevant fibrillar collagen I matrices, which are highly non-elastic, cells exhibit additional modes of cell volume-dependent mechanosensing that are not observable on elastic substrates. Notably, volumetric compression regulates the dynamics of cell-ECM interactions and irreversible ECM remodeling via Rac-directed protrusion dynamics, at both the single-cell level and the multicellular level. Our findings support that cell volume is a master biophysical regulator and reveal its roles in cell mechanical state transition, cell-ECM interactions, and biophysical tissue programming.

biophysics↗

bioRxiv2023

Adaptation to volumetric compression drives hepatoblastoma cells to an apoptosis-resistant and invasive phenotype

Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and {beta}-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells. Significance statementCompression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.

cancer biology↗

bioRxiv2023

Instant Assembly of Collagen for Scaffolding, Tissue Engineering, and Bioprinting

Controllable assembly of cells and tissues offers potential for advancing disease and development modeling and regenerative medicine. The bodys natural scaffolding material is the extracellular matrix, composed largely of collagen I. However, challenges in precisely controlling collagen assembly limit collagens applicability as a primary bioink or glue for biofabrication. Here, we introduce a set of biopatterning methods, termed Tunable Rapid Assembly of Collagenous Elements (TRACE), that enables instant gelation and rapid patterning of collagen I solutions with wide range of concentrations. Our methods are based on accelerating the gelation of collagen solutions to instantaneous speeds via macromolecular crowding, allowing versatile patterning of both cell-free and cell-laden collagen-based bioinks. We demonstrate notable applications, including macroscopic organoid engineering, rapid free-form 3D bioprinting, contractile cardiac ventricle model, and patterning of high-resolution (below 5 (m) collagen filament. Our findings enable more controllable and versatile applications for multi-scale collagen-based biofabrication.

bioengineering↗

bioRxiv2023

Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements severely limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) Inhibitor-induced changes of traction forces and fiber alignment and density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

biophysics↗

bioRxiv2018

Cytoskeletal tension actively sustains the T cell immunological synapse

When migratory T cells encounter antigen presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T cell immunity. While the cellular processes underlying synapse formation have been well-characterized, those that maintain the symmetry, and thereby the stability of the synapse remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott -Aldrich syndrome Protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T cell activation, WASP is degraded, leading to cytoskeletal rearrangement and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

cell biology↗

bioRxiv2017

Non-Elastic Remodeling of the 3D Extracellular Matrix by Cell-Generated Forces

The mechanical properties of the extracellular matrix (ECM) - a complex, 3D, fibrillar scaffold of cells in physiological environments - modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local environments. Here, we demonstrate that cell generated contractile forces are capable of producing substantial irreversible changes to the density and architecture of physiologically relevant ECMs - collagen I and fibrin - in a matter of minutes. We measure the 3D mechanical deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale modeling. Our findings reveal that plasticity, as a mechanical law in these networks, is fundamentally related to the force-driven unbinding of fiber crosslinks. These results illustrate the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems.

bioengineering↗