The Lomakin Lab
Background information: Our previous efforts have led to the discovery of the nucleus as a critical cellular component capable of monitoring mechanical stress and converting this injury input into signaling and metabolic 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 noted that enucleated cytoplasts can still detect and respond to mechanical insults (albeit of a stronger magnitude) by changing the organization of their cytoplasm and whole-cell morphodynamics. This suggested that cells, in addition to the nucleus, can employ other cellular 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).
Recent comparative transcriptomics analyses of various tissue types from mammalian species with diverse lifespans, have revealed that, in addition to DNA repair and RNA transport pathways traditionally associated with aging and longevity, the mechanisms controlling MT organization are positively correlated with a species’ maximum lifespan (Fig. 2). The general trend is that short-lived species exhibit decaying MT networks, while long-lived species possess robust MT arrays (Lu et al. Cell Metabolism, 2022). Accordingly, MT-damaging pharmacological agents downregulate the genes that enable a species’ maximum lifespan, highlighting that MT stability and organization are causative factors in longevity. These new data suggest that MTs can be considered emerging regulators of both aging and longevity.
Figure 2 (adapted from Lu et al. Cell Metabolism, 2022).
Addressed problem: 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. One potentially promising approach 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 primary research goal.
Specific questions:
1) 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 phenotype of therapy-induced senescence (TIS) (Demaria et al. Cancer Discovery, 2017). One of the most universally observable traits of such cells is cell size inflation and poly(aneu)ploidy triggered by abnormalities in the functioning of the microtubule (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. 3). 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 cell size change remains completely unknown.
Figure 3 (prepared partially using the BIORender App).
2) Various mammalian species with distinct lifespans and aging rates seem to exhibit different degrees of MT system stability (Lu et al. Cell Metabolism, 2022 and Fig. 2) and intrinsically distinct sizes of tissue cells including neurons (Beaulieu-Laroche et al. Nature, 2021 and Fig. 4), where MTs are of paramount importance to cell morphology and physiology. We therefore hypothesize that the MT system and the MIR program are evolutionarily tuned in distinct mammalian species, including humans and nonhuman primates, to coordinate tissue cell morphology and growth with gene expression pace, ultimately predetermining developmental and aging rates. These rates vary significantly among different mammals (Lázaro et al. Cell Stem Cell, 2023). Indeed, Ernst Haeckel documented this in his Natürliche Schöpfungsgeschichte back in 1868. However, the cellular control mechanisms behind this phenomenon remain poorly understood. We believe that quantitatively comparing and experimentally perturbing MT dynamics and mechanics across species might reveal new insights into the control of cellular growth pace and aging.
Figure 4 (adapted from Beaulieu-Laroche et al. Nature, 2021).
Very broadly speaking, this kind of research might not only impact the fields of cell biology, biophysics, aging, and cancer research but also evolutionary biology. It appears that cell size increases from simpler organisms like E. coli to more complex organisms such as H. sapiens. As species become more complex through evolution, there is a need for more intricate and robust cytoskeletal structures to support larger cells and more complex cellular functions. This highlights a co-evolutionary trend where increases in cell size are associated with increased complexity of the tubulin MT system (Fig. 5). However, it is not clear whether the increased complexity of the MT system is a response to cell size increase in evolution, or if the evolved complexity of the MT system dictates cell size increase. Answering this question is an exciting direction for future studies.
Figure 5 (prepared using the BIORender App).
Approach: We employ a scale-bridging approach (Fig. 6) 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 natural or taxane chemotherapy-triggered aging-associated 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 mechanical 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 examine the generality of the principles we uncover by performing comparative cell biology studies. We take advantage of the fact that tissue cells derived from distinct mammalian species exhibit intrinsically different cell sizes, enabling the use of species-specific cell lines as a library of naturally evolved tissue cell sizes. Our approaches and projects will interest scholars focused on quantitative cell biology and biophysics, microscopy and bioimage informatics, pharmacobiology, drug resistance mechanisms, cancer and aging research, and evolutionary and comparative cell biology.
Figure 6 (partially adapted from Kabakova et al. Nature Reviews Methods Primers, 2024 and Nagy et al. eLife, 2024).
Medical University of Vienna
Center for Pathobiochemistry and Genetics
