Biology subjects

Stecker, K. E.

Publications and source records attributed to Stecker, K. E..

13 recordsLinked to original sources

bioRxiv2026

TSPAN3 defines a distinct intracellular trafficking route to secretory multivesicular bodies

Extracellular vesicles (EVs) comprise molecularly diverse populations generated through multiple membrane-trafficking pathways, yet the intracellular basis of this heterogeneity remains poorly understood. Here, we identify the EV-associated tetraspanin TSPAN3 as a marker of a secretory multivesicular body (MVB) population that is molecularly and functionally distinct from canonical CD63-positive compartments. Using endogenous genome editing, live-cell and super-resolution microscopy, electron microscopy, quantitative EV secretion assays, and complementary proteomic approaches, we show that TSPAN3 localizes to fusion-competent MVBs but exhibits limited overlap with CD63 during secretion. Unlike CD63, which extensively traffics through the plasma membrane and depends on YXX{Phi}-mediated endocytic retrieval, TSPAN3 reaches secretory MVBs predominantly through an intracellular trafficking route that relies on a dileucine-containing sorting region. Orthogonal proximity-labeling and affinity-purification proteomics revealed that TSPAN3-positive compartments are associated with a selective LC3/ATG8-related membrane network, including GABARAPL2 and proteins involved in endosomal membrane remodeling and fusion. Perturbation of residues required for this association impaired localization to LC3-positive compartments and reduced secretory MVB fusion. Consistent with these findings, pharmacological disruption of autophagy- and endolysosomal-associated pathways differentially altered TSPAN3-positive EV secretion. Finally, proximity-labeled EV proteomics demonstrated that TSPAN3-associated EVs possess cargo signatures distinct from CD63-associated EVs, with greater representation of endosomal and endolysosomal proteins suggesting that tetraspanin-associated membrane nanodomains retain molecular signatures consistent with their intracellular trafficking history. Together, our findings identify TSPAN3 as a marker of a previously unrecognized secretory MVB population distinguished by its intracellular trafficking, molecular interactions, and EV composition, supporting a model in which distinct tetraspanin-organized membrane nanodomains are associated with different intracellular trafficking routes and molecularly distinct EV populations.

cell biology↗

bioRxiv2026

Dissection of centrosomal γ-TuRC activation pathways controlling microtubule density in interphase cells

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.

cell biology↗

bioRxiv2026

Regulation of microtubule abundance and minus end dynamics by Katanin, CAMSAPs, WDR47 and kinesin-13

Microtubule networks are major determinants of cell architecture and logistics. Microtubule organization and density are regulated by severing enzymes, which cut microtubule lattices or affect their growth and shortening. These activities can lead to microtubule amplification or disassembly, depending on the presence of microtubule stabilizers or destabilizers, but the interplay between these factors is poorly understood. Here, we reconstituted in vitro the activity of microtubule severase katanin together with microtubule minus-end stabilizers CAMSAPs, their binding partner WDR47 and microtubule depolymerase kinesin-13/MCAK. We confirmed that katanin can amplify or destroy microtubules in a concentration-dependent manner. CAMSAPs recruit katanin to microtubules and reduce katanin concentration needed for both amplification and destruction, whereas kinesin-13 completely abolishes microtubule amplification. WDR47 binds to microtubules decorated by CAMSAPs and suppresses katanin binding and severing. In addition, both katanin and WDR47 inhibit polymerization of CAMSAP-decorated microtubule minus ends. These data explain how these proteins act together to fine-tune microtubule minus-end stability without strongly increasing microtubule abundance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/714132v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@746fe3org.highwire.dtl.DTLVardef@5dd5a8org.highwire.dtl.DTLVardef@762373org.highwire.dtl.DTLVardef@1192db_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cell biology↗

bioRxiv2025

Taxane-Induced Conformational Changes in the Microtubule Lattice Activate GEF-H1-Dependent RhoA Signaling

Taxanes are widely used chemotherapeutic agents that perturb cell division. They also exert effects during interphase, but the underlying mechanisms are poorly understood. Here, we show that taxanes activate RhoA signaling and induce actin remodeling by displacing the RhoA activator GEF-H1 from microtubules. This taxane-induced release of GEF-H1 occurs rapidly, is independent of tubulin post-translational modifications, and can be recapitulated using purified proteins. In vitro reconstitution assays combined with analyses of microtubule structure revealed that microtubule binding by GEF-H1 is inhibited by microtubule-stabilizing agents that expand the microtubule lattice, such as taxanes and GMPCPP, but not by others, including GTP{gamma}S and discodermolide, which stabilize a compacted microtubule lattice. Our findings demonstrate that alterations in microtubule lattice conformation can activate key signaling pathways, offering new insights into the mode of action of taxanes and the possible origins of their side effects.

cell biology↗

bioRxiv2025

EVtrace: tracing extracellular vesicles-associated proteins in recipient cells using stable isotope labeling

Release and uptake of extracellular vesicles (EVs) is a highly conserved means of communication used by cells across all kingdoms of life. These nano-sized vesicles transfer messages encoded in proteins and nucleic acids and play a role in numerous (patho)physiological processes. A longstanding question in the field is whether interaction and uptake of EVs is a stochastic process or whether specific EV subpopulations target different types of recipient cells. Here we present EVtrace, a proteomics-based approach that accommodates stable isotopic labeling of amino acids in culture (SILAC) to trace back labeled EV-associated proteins in unlabeled recipient cell types. We describe the optimization of EV labeling conditions and EV-cell cultures, and introduce a proteomics data analysis pipeline to confidently identify sparse internalized EV proteins among unlabeled recipient cell proteins. As a proof of concept, we studied the targeting of prostate cancer (PCa) EVs to bone cells and non-bone cells, considering that bone is a common metastatic site of PCa. Using EVtrace we demonstrate that interaction of PCa EVs with recipient cell types was not stochastic, and that different subpopulations of EVs targeted different cells. Proteins of EV subpopulations that were traced in bone cells, but not in non-bone cells, were strongly enriched for pathways involved in cancer progression and metastasis. With super-resolution microscopy we confirmed that EV proteins traced in different targeted cell types co-occurred on specific EV subpopulations. EVtrace is a valuable new tool for EV research because it supports identification of EV subpopulations that are transferred to recipient cells and discloses candidate proteins potentially involved in EV binding/uptake and functional modification of target cells.

molecular biology↗

bioRxiv2025

GDF15 regulates necroptotic cell death through direct interaction with RIPK3

Enhancing the immunogenicity of tumor cells is a major objective in cancer therapy, particularly for tumors with low immune cell infiltration scores. Inducing immunogenic forms of programmed cell death (PCD) offers a promising strategy to strengthen anti-tumor immunity and improve therapeutic outcomes. Necroptosis, a highly inflammatory form of regulated cell death triggered by TNF signaling, can elicit robust immune activation. However, its regulation in tumor cells remains incompletely understood, limiting its therapeutic exploitation. To investigate the protein-protein interactions that govern necroptotic cell death in tumor cells, we established a co-Immunoprecipitation - Mass Spectrometry (coIP-MS) workflow using RIPK3, the central effector kinase driving necroptosis in TNF-induced signaling, as bait. This unbiased proteomic approach enables the identification of candidate regulators directly associated with the necrosome complex components under active necroptotic conditions. Among identified candidates, TCOF1 and GDF15 emerged as previously unrecognized modulators, with functional knockout of either gene markedly enhancing necroptotic cell death in tumor cells. Reciprocal IP experiments confirmed a direct interaction between GDF15 and RIPK3, supporting its mechanistic role as a negative regulator that suppresses necroptotic signaling. Thus, our findings extend the function of GDF15 beyond its established role in inflammation, uncovering an additional layer of regulation at the level of cell-intrinsic death signaling. Collectively, our findings position GDF15 as a RIPK3-interacting "brake" on necroptotic cell death and highlight TCOF1 as an additional inhibitory node. Our study underscores the potential of targeting necroptosis-suppressive mechanisms to influence PCD outcomes in tumors and demonstrates the power of coIP-MS for mapping TNF-induced interactions to reveal actionable molecular targets for tumor sensitization.

cell biology↗

bioRxiv2025

Decoding interaction-induced proteome changes in co-cultures with hybrid quantification and SILAC-directed real-time search

Intercellular communication between T cells and cancer cells plays a pivotal role in determining cancer cell survival or death. Yet, our understanding of this interaction remains incomplete. Methods to study heterotypic cell interactions are either limited to targeted studies relying on predefined set of proteins, or require cell separation, thus disrupting the native environment. Stable isotope labeling by amino acids in cell culture (SILAC) enables proteome distinction in heterologous co-cultures without the need for physical separation. But dynamic studies remain constrained by the need for numerous mass spectrometry (MS) runs, the challenges in detecting low-abundant proteins, particularly in immune cells and the limited data completeness due to the use of data-dependent MS1-based precursor quantification. To overcome these limitations, we evaluate the integration of SILAC with tandem mass tag (TMT) multiplexing and SILAC-directed real-time search (RTS). TMT labeling enables simultaneous analysis of multiple samples, while RTS-MS3 acquisition using SILAC-induced mass shifts as fixed modifications triggers MS3 scans for specific proteome populations within a mixed cell system, improving quantitative accuracy and proteome coverage for target protein populations. We benchmarked our acquisition methods using SILAC-labeled samples mixed at defined ratios and validated the approach in biologically relevant co-culture experiments. Additionally, we introduced a carrier channel to enhance detection of lower-abundant T cell proteins, while maintaining acceptable quantitative precision. Our results demonstrate that the combined SILAC-TMT-RTS strategy dramatically improves proteome depth, temporal resolution, and cell-type specificity for short-term co-culture interaction proteomics studies. In co-culture samples of T cells with non-small cell lung cancer cell lines that were either sensitive or resistant to T cell killing, our method revealed candidate mechanisms underlying their differential sensitivity. Our integrated approach combining SILAC, TMT and RTS to resolve cell-specific proteome dynamics in co-culture represents a novel and powerful advance.

cancer biology↗

bioRxiv2025

Inhibition of TBK1/IKKe mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing

Resistance to immune cell-mediated cytotoxicity poses a significant challenge in cancer therapy, compromising the efficacy of immunotherapeutic approaches such as immune checkpoint blockade (ICB) treatment. To enhance therapy outcomes, it is crucial to identify interventions that can synergize with ICB therapy to overcome tumor resistance. Therefore, we need to define the cellular mechanisms that sensitize tumors to cytotoxic T cells. CD8 T cells rely on cytokines such as TNF to carry out their cytotoxicity against tumors, and recent findings link select tumor mutations in the TNF pathway to increased T cell killing, in a manner dependent on RIPK1 kinase. Here, we demonstrate that sensitized tumor cells fail to initiate inhibitory RIPK1 phosphorylation at site S25 upon T cell attack, thereby foregoing a pro-survival checkpoint early in TNF signal transduction. Consequently, tumor cells experiencing a loss of TNF-induced RIPK1 S25 phosphorylation exhibit increased RIPK1 activation and fail to recruit non-canonical IKK kinases (TBK1 and IKKe) to the TNFR1 complex. Functional knockouts of TBK1 and IKKe in melanoma cells result in heightened sensitivity not only in CD8 T cell but also in Natural Killer cell attacks. Our findings indicate that preventing TBK1 and IKKe recruitment to the TNF signaling complex, thereby blocking RIPK1 pro-survival phosphorylation and promoting direct RIPK1 activation, is a tractable strategy to increase tumor sensitivity to immune cell killing and has the potential to benefit current immunotherapy interventions.

cell biology↗

bioRxiv2024

Probing DNA damage sites reveals context-dependent and novel DNA damage response factors

DNA damage is a constant threat to genome integrity and function. Diminished capacity for DNA repair is linked to many human diseases, therefore understanding the molecular pathways responding to DNA damage is key for developing novel therapies. Lack of unbiased probes to report DNA damage dynamics and the associated proteins in living cells and animals limit our current efforts to completely understand DNA repair processes. In this study we overcome these limitations by engineering protein probes containing the tandem-BRCT domain of MCPH1, which we show to have a specific affinity for the DNA-damage-associated histone mark {gamma}H2AX. We employ these probes to track DNA damage dynamics in living cells exposed to a panel of different genotoxic insults and to visualize programmed double strand breaks during gametogenesis in living animals. We further utilize the binding selectivity of our probe to tether TurboID biotin ligases to chromatin and identify the DNA damage-associated proteome via proximity ligation. By comparing five different DNA damaging agents, we reveal the proteome associated with specific lesions, and identify multiple novel proteins with potential implications in damage response and repair. Among these novel proteins, we characterize the ubiquitin ligase UBE3A, the methyl-binding and proteasome-recruiting protein L3MBTL3, and the spliceosomal factor U2SURP, as previously uncharacterized effectors of DNA damage response. These functional datasets reveal the DNA damage-dependent proteomes and reveal novel insights into DNA damage response.

molecular biology↗

bioRxiv2024

A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Cilia are essential motile or sensory organelles found on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, however the underlying biochemical mechanisms are largely unexplored. Here, we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins, CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules, and, when mutated, cause ciliopathies such as Joubert syndrome. CEP104, a protein containing a tubulin-binding TOG domain, is an inhibitor of microtubule growth and shortening that interacts with EBs on the microtubule surface and with a luminal microtubule-pausing factor CSPP1. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics directly but act as scaffolds for their partners. Cryo-electron tomography showed that together, ciliary tip module members form plus-end-specific cork-like structures which reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

cell biology↗

bioRxiv2023

Phosphoprotein dynamics of interacting tumor and T cells by HySic

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. Some of the proteins involved, particularly immune checkpoints expressed by T cells, serve as promising clinical targets in immunotherapy. However, our understanding of the complexity and dynamics of the interactions between tumor cells and T cells is only rudimentary. Here we present HySic (for Hybrid quantification of SILAC (Stable Isotope Labelling by Amino acids in Cell culture)-labeled interacting cells) as an innovative method to quantify protein and phosphorylation dynamics between and within physically interacting (heterotypic) cells. We show that co-cultured HLA/antigen-matched tumor and T cells engage in physical and stable interactions, allowing for in-depth HySic analysis. This method does not require physical separation of the two cell types for subsequent MS proteome and phosphoproteome measurement using label free quantification (LFQ). We demonstrate that HySic can be used to unravel proteins contributing to functional T cell:tumor cell interactions. We validated HySic with established interactions, including those mediating IFN{gamma} signaling. Using HySic we identified the RHO/RAC/PAK1 signaling pathway to be activated upon interaction of T cells and tumor cells. Pharmacologic inhibition of PAK1 sensitized tumor cells to T cell killing. Thus, HySic is an innovative and simple method to study short-term protein signaling dynamics in physically interacting cells, which can be easily extended to other biological systems.

cancer biology↗

bioRxiv2022

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates elongation and stability of ciliary axonemes and other microtubule-based structures.

cell biology↗

bioRxiv2021

Microtubule plus-end regulation by centriolar cap proteins

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS- 4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus- end dynamics and centriole biogenesis.

cell biology↗