The Lomakin Lab
Background information: Our previous efforts have led to the discovery of the nucleus as a critical subcellular domain capable of monitoring mechanical environmental stress and converting this injury input into signaling and metabolic molecular outputs, thus reprogramming cell form and growth in a nongenetic fashion (Lomakin et al. Science, 2020). However, when we engineered live cells without the nucleus (cytoplasts) and mechanically challenged them, we observed that enucleated cytoplasts can still detect and respond to mechanical insults (albeit of a stronger magnitude) by altering mechanochemical reactions at the submembranous cortex. This, in turn, triggers a spontaneous switch in cytoplast morphology and dynamics, conferring an adaptive advantage under high mechanical environmental stress. These intriguing observations suggested that cells, in addition to the nucleus, can employ other cellular/molecular components to detect stress stimuli. Given the crucial role of cytoplasmic microtubules (MTs) in the integrated stress response (ISR) triggered by oxidative damage, as previously described by us (Nadezhdina and Lomakin et al. BBA, 2010), we hypothesized that these subcellular components might be involved in cellular stress response in other contexts as well. Indeed, we recently contributed to the discovery of MTs as critical sensors of biophysical stress, allowing cells to survive mechanical injuries while maneuvering through spatially confining microenvironments (Ju et al. Nature Cell Biology, 2024; see Highlight by Lisa Heinke in Nature Reviews Molecular Cell Biology and Dispatch by Anna Akhmanova & Manuel Thery in Current Biology).
So, what are MTs, and why are they relevant in the context of stress response?
MTs, the semiflexible polymers of the cytoskeletal GTPase protein tubulin, serve as supramolecular highways for intracellular transport. Fueled by the energy from ATP hydrolysis, the mechanochemical nanomachines dynein and kinesin travel along MTs, transporting intracellular cargoes to various subcellular destinations (https://youtu.be/y-uuk4Pr2i8?si=mfVOF4-S6J0Kv-Fg). The dynamic instability of MTs, characterized by stochastic phases of polymer assembly and disassembly (https://youtu.be/tO-W8mvBa78?si=T3ECjPvcrUuSB2sv), along with their organization into larger-scale networks, is fundamental to the trafficking of cytoplasmic vesicles and macromolecular complexes in interphase cells, as well as nuclear genome partitioning during mitosis (https://youtu.be/IvJrDsRuWxQ?si=hIWkR-K4HQrJDY5a). Similar to macroscopic objects in both the living and nonliving worlds, cellular MTs are subject to mechanical damage, chemical poisoning, and aging (Fig. 1). Structural and regulatory proteins associated with the MT cytoskeleton monitor these insults and activate the "Microtubule Integrity Response" (MIR) (Gasic et al. PLoS Biology, 2019) as a defense mechanism. While the molecular signaling and physico-chemical mechanisms underlying this recently proposed stress response program are still poorly understood (Gasic & Mitchison. Current Opinion in Cell Biology, 2019; Lin et al. Science, 2019; and Höpfler et al. Molecular Cell, 2023), it is evident that they fail in some circumstances. Indeed, MT wear and tear are strongly associated with neuronal aging and neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases (Okenve-Ramos et al. PLoS Biology, 2024). Moreover, MT system malfunction contributes to increased infertility of female oocytes with age (Nakagawa & FitzHarris. Current Biology, 2017) and drives inflammaging of somatic tissues due to the accumulation of aneuploid cells with a proinflammatory senescence-associated secretory phenotype (Barroso‐Vilares et al. EMBO reports, 2020).
Figure 1 (prepared using the BIORender App).
Addressed problem: While our previous studies were concentrated primarily on crucial cytoskeletal elements, including MTs, in the context of mechanical environmental stress sensing and phenotypic resistance, our current focus is on chemical (drug-induced) stress sensing and adaptations. Drugs targeting tubulin assembly-disassembly or MT network organization may represent a previously unrecognized class of aging-modifying compounds. However, the field of oncobiology has long embraced tubulin and MTs as classic molecular targets for anti-cancer drugs (Dumontet & Jordan. Nature Reviews Drug Discovery, 2010) revealing numerous and even life-threatening side effects of such compounds. This challenges their clinical utility in the aging and longevity domain. Moreover, as with nearly all chemotherapeutics used in the clinical oncology domain, resistance inevitably emerges to MT-targeting drugs. One potentially promising solution to these problems could involve modifying the MIR mechanism rather than directly targeting the MT system itself. Enhancing MIR in the context of biological aging or disrupting it in the context of cancer could offer unprecedented new therapeutic avenues. To achieve this translational goal, it is crucial to first acquire a fundamental understanding of how MTs sense damage inputs, evoke the MIR program, and generate cellular behavioral outputs, including adaptations in morphology, growth, and metabolism that ultimately increase cell fitness in the face of stress. Addressing these questions is our main research goal.
Specific question:
Focusing on the blockbuster oncology drugs of the Taxane family (Weaver. Molecular Biology of the Cell, 2017) that target MT mechanics and dynamics (Flemming. Nature Reviews Drug Discovery, 2017 and Rai et al. Nature Materials, 2020), we wish to discover the quantitative molecular and physico-chemical mechanisms by which the subtle damage caused to the MT system by these drugs at clinically relevant non-saturating concentrations (Castle et al. Molecular Biology of the Cell, 2017) is sensed and translated via the MIR program into complex cell behaviors including the phenotypic switch to therapy-induced senescence (TIS) (Demaria et al. Cancer Discovery, 2017). A hallmark of these cells is enlarged size and poly(aneu)ploidy (the so-called Titan cell phenotype), triggered by abnormalities in the functioning of the MT system (Belhadj et al. Aging Cell, 2023). As a result, human cancer cells experiencing TIS begin to engage transcriptional and signaling programs associated with cell size control (Fig. 2). This, in turn, suggests that MT-mediated cell size regulation mechanisms are a vulnerability that can be exploited therapeutically to overcome drug resistance and improve the outcomes of current chemotherapy regimens in treating various epithelial cancers, including breast and prostate carcinomas. However, how and why MT damage in cells under sublethal chemotherapeutic stress is converted into a Titan(-like) phenotype remains to be fully elucidated.
Figure 2 (prepared partially using the BIORender App).
Approach: We employ a scale-bridging approach (Fig. 3) that links cell size and content phenotypic readouts (based on physico-chemical methods such as buoyant density (mass over volume) estimation using isopycnic centrifugation of cells, Brillouin spectroscopy to determine water content of cells, genetically-encoded inert cytoplasmic probes for cell viscosity measurements based on single-particle tracking algorithms, and AI-driven quantitative microscopy to measure cell morphometric features) to the global molecular profile of cells (obtained via RNA-seq, proteomics, and metabolomics). This holistic approach aims to understand how cells allocate their molecular resources during Taxane stress-triggered cell size alterations. Relying on these quantitative phenotypic and molecular readouts (morpho-molecular phenotyping), we build phenomenological and mechanistic biophysical models to forecast scaling relationships between molecular pools, organellar sizes, and whole-cell size. We test these models using cell engineering approaches (e.g., by generating live mini-cells without the nucleus or molding cells into different shapes and sizes via micropatterning-microfabrication) and targeted molecular perturbations. Finally, we assess the generality of the mechanisms we uncover by conducting comparative drug response studies across evolutionarily distant species. To this end, we use genetically engineered strains of
Saccharomyces cerevisiae expressing "humanized" tubulin, which renders them sensitive to Taxanes (Fig. 4).
Figure 3 (partially adapted from Kabakova et al. Nature Reviews Methods Primers, 2024 and Nagy et al. eLife, 2024).
Figure 4 (MDR-deficient -/+ Taxane sensitive strains of S. cerevisiae, Lomakin Lab).
Our approaches and projects will interest scholars focused on quantitative cell biology and biophysics, bioinformatics, microscopy and bioimage informatics, pharmacobiology, drug resistance mechanisms, cancer & aging research, and comparative cell biology.
Medical University of Vienna
Center for Pathobiochemistry and Genetics
