LABORATORY OF CELL MOTILITY
Laboratory of Cell Motility focuses on analysis of cell behavior and role of cytoskeleton in motility and proliferation of normal and tumor cells. We are interested in understanding, at a systems level, how specific features of cytoskeleton are coordinated with cell dynamics. Our studies using cultured normal and cancer mammalian cells are related to the development of anti-cancer therapy programs by in-depth analysis of cell behavior and proliferation in respect to different treatments.
We study different mitotic inhibitors, i.e. drugs stabilizing or depolymerizing microtubules, inhibitors of actin cytoskeleton and myosin and behavior of focal contacts – specific structures responsible for attachment of cell to the substrate and motility of cells on the solid substrates.
We implement high-throughput time lapse microscopy as well as different types of confocal microscopy analyzing living and fixed cells. Together with computer specialists we are developing image analysis tools for high-throughput analysis of cytoskeleton organization and cell motility. Other methods include but are not limited to flow cytometry (with imaging flow cytometry and spectral cytometry), knockdown technologies of different cytoskeletal components.
RESEARCH AREAS
1. Cytoskeleton changes in cells after EMT
Actin filaments(shown in red) MCF-7 cells after EMT,where the GFP nucleus staining(shown in green)
Fluorescence microscopy analysis of actin cytoskeleton rearrangements in MCF-7, A-549 and HaCaT cells after EMT. (a) Staining with Alexa Fluor 555-labeled phalloidin. Scale bar 10 µm. (b) Actin stress fiber count in three different cell lines before and after EMT. (c) Angular distribution of actin filaments (σ) in cells before and after EMT.
We induced epithelial-to-mesenchymal transition (EMT) in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We analyzed dynamics of microtubules and focal adhesions, and spatial organization of stress fibers. The following changes were observed after EMT: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules.
We plan to assess the expression profile of actin and tubulin isoforms during induction of EMT in tumor cells and respective changes in the cytoskeleton behavior.
Find out more: Nurmagambetova, A., Mustyatsa, V., Saidova, A. et al. Morphological and cytoskeleton changes in cells after EMT. Sci Rep 13, 22164 (2023). https://doi.org/10.1038/s41598-023-48279-y
We plan to assess the expression profile of actin and tubulin isoforms during induction of EMT in tumor cells and respective changes in the cytoskeleton behavior.
Find out more: Nurmagambetova, A., Mustyatsa, V., Saidova, A. et al. Morphological and cytoskeleton changes in cells after EMT. Sci Rep 13, 22164 (2023). https://doi.org/10.1038/s41598-023-48279-y
2. Focal adhesions in mobile cells
The project is devoted to the study of the organization of focal adhesions (FAs) and their role in cell movement. FAs were described more than 50 years ago, but their role in cells remains unclear and ideas about the dynamics are contradictory. FAs are complexes of proteins that are assembled on the cell membrane during its interaction with the substrate and transmit mechanical tension and chemical signals from the cell surface. The formation of FAs is necessary for cell spreading and movement along the substrate.
We evaluated FA dynamics by time-lapse microscopy with high spatial and temporal resolution, tracking FA size, lifetime, and intensity in A549 cells stably expressing Vinculin-RFP. We observed numerous small, short-living adhesions at the leading edge, occurring both at large lamellae aligning with the vector of cell body translocation and smaller lateral lamellae. That contrasted with the presence of only a few large adhesions at the trailing edge. Most FAs present at the trailing edge originated within lateral protrusions and only few of them formed directly there. During retraction of the rear cell edge, a portion of FAs has already been disassembled before retraction has reached them. However, some FAs at the trailing edge were enlarging, either through merging with neighboring FAs or gradual increase in area and. As a result, relatively large FAs were forcibly detached and rapidly disassembled only upon retraction edge reached them.
In the context of coordinated cell migration, discernible disparity between the dynamics of FAs emerges across different cell regions, where the lot of 'lilliputians' at the leading edge ultimately defeat the 'Gullivers' at the trailing edge.
The maturation of FAs continues through the involvement of focal adhesion kinase (FAK) and the mechanosensory proteins talin and vinculin, which provide formation of a mature FA structure and its association with actin microfilaments. FAK is a tyrosine kinase that becomes activated within FA, where it regulates cell adhesion, motility, and, probably, cell survival. FAK is one of the first specific proteins that is concentrated in FA and builds it through phosphorylation of specific proteins. FAK is often overexpressed in tumor cells, and can be a target for anticancer therapy. In the current project detailed studies of the role of FAK in the organization of FAs and cell motility will be performed.
In further studies we are going to test
hypothesis that FAK mediates the rapid formation and maturation of focal adhesion and associated cytoskeletal rearrangement playing central role in cell attachment, spreading, and motility.
Find out more: American Society for Cell Biology. 2023 ASCB Annual Meeting Abstracts. Mol Biol Cell. 2024 Jan 1;35(1):ar12. doi: 10.1091/mbc.E23-10-0402. PMID: 38108669; PMCID: PMC10881173.
We evaluated FA dynamics by time-lapse microscopy with high spatial and temporal resolution, tracking FA size, lifetime, and intensity in A549 cells stably expressing Vinculin-RFP. We observed numerous small, short-living adhesions at the leading edge, occurring both at large lamellae aligning with the vector of cell body translocation and smaller lateral lamellae. That contrasted with the presence of only a few large adhesions at the trailing edge. Most FAs present at the trailing edge originated within lateral protrusions and only few of them formed directly there. During retraction of the rear cell edge, a portion of FAs has already been disassembled before retraction has reached them. However, some FAs at the trailing edge were enlarging, either through merging with neighboring FAs or gradual increase in area and. As a result, relatively large FAs were forcibly detached and rapidly disassembled only upon retraction edge reached them.
In the context of coordinated cell migration, discernible disparity between the dynamics of FAs emerges across different cell regions, where the lot of 'lilliputians' at the leading edge ultimately defeat the 'Gullivers' at the trailing edge.
The maturation of FAs continues through the involvement of focal adhesion kinase (FAK) and the mechanosensory proteins talin and vinculin, which provide formation of a mature FA structure and its association with actin microfilaments. FAK is a tyrosine kinase that becomes activated within FA, where it regulates cell adhesion, motility, and, probably, cell survival. FAK is one of the first specific proteins that is concentrated in FA and builds it through phosphorylation of specific proteins. FAK is often overexpressed in tumor cells, and can be a target for anticancer therapy. In the current project detailed studies of the role of FAK in the organization of FAs and cell motility will be performed.
In further studies we are going to test
hypothesis that FAK mediates the rapid formation and maturation of focal adhesion and associated cytoskeletal rearrangement playing central role in cell attachment, spreading, and motility.
Find out more: American Society for Cell Biology. 2023 ASCB Annual Meeting Abstracts. Mol Biol Cell. 2024 Jan 1;35(1):ar12. doi: 10.1091/mbc.E23-10-0402. PMID: 38108669; PMCID: PMC10881173.
3. Regulation of outcomes and consequences of mitotic arrest
Histograms showing cell distribution of HeLa cells according to DNA content after 48 h treatment with anti-microtubule drugs. Minimal threshold concentrations (T1) for each drug are indicated by long arrow, and maximal threshold concentrations (T2)—by arrowhead.
Time-lapse sequences illustrating three fates (mitotic slippage, death after mitotic slippage, and death in mitosis) exhibited by A549 cells under the treatment with 1 uM Taxol. Numbers indicate time from the moment of mitotic entry in hours. Mitochondrial membrane potential (red), active caspases 3/7 (green), and nuclei (blue) are indicated. Scale bar = 20 µm.
Microtubule-targeting (MT) drugs taxanes and vinca alkaloids are widely used as chemotherapeutic agents against different tumors for more than 30 years because of their ability to block mitotic progression by disrupting the mitotic spindle and activating the spindle assembly checkpoint (SAC) for a prolonged period of time. However, responses to mitotic arrest are different—some cells die during mitotic arrest, whereas others undergo mitotic slippage and survive becoming able for proliferation. Using normal fibroblasts and several cancer cell types we determined two critical doses, T1 and T2, of mitotic inhibitors (nocodazole, Taxol, and vinorelbine). T1 is the maximal dose cells can tolerate undergoing normal division, and T2 is the minimal mitostatic dose, wherein majority of mitotic cells are arrested in mitosis. In all studied cell lines after treatment with mitotic inhibitors in a dose above T2 cells had entered mitosis either die or undergo mitotic slippage. We show that for all three drugs used cell death during mitotic arrest and after slippage proceeded via mitochondria-dependent apoptosis. We determined two types of cancer cells: sensitive to mitotic arrest, that is, undergoing death in mitosis (DiM) frequently, and resistant to mitotic arrest, that is, undergoing mitotic slippage followed by prolonged survival. We then determined that inhibition of Bcl-xL, but not other anti-apoptotic proteins of the Bcl-2 group that regulate apoptosis, make resistant cells susceptible to DiM induced by mitotic inhibitors. Combined treatment with MT drugs and highly specific Bcl-xL inhibitors A-1155643 or A-1331852 allows achieving 100% death in mitosis in a time significantly shorter than maximal duration of mitotic arrest. We further examined efficacy of sequential treatment of cultured cells using mitotic inhibitors followed by inhibitors of Bcl-xL anti-apoptotic protein and for the first time show that sensitivity to Bcl-xL inhibitors rapidly declines after mitotic slippage. Thus sequential use of mitotic inhibitors and inhibitors of Bcl-xL anti-apoptotic protein will be efficient only if the Bcl-xL inhibitor will be added before mitotic slippage occurs or soon afterward. The combined treatment proposed might be an efficient approach to anti-cancer therapy.
We currently are analyzing long-term consequences of mitotic arrest after washout of microtubule-stabilizing drugs. The initial event of relatively short treatment with microtubule stabilizers of cell cultures prone to mitotic slippage is formation of the numerous multi-nucleated cells. Restoration of normal divisions takes long time. We address the question what cells are the source of delayed recovery after drug washout.
Find out more: Suleimenov M, Bekbayev S, Ten M, Suleimenova N, Tlegenova M, Nurmagambetova A, Kauanova S and Vorobjev I (2022) Bcl-xL activity influences outcome of the mitotic arrest. Front. Pharmacol. 13:933112. doi: 10.3389/fphar.2022.933112
We currently are analyzing long-term consequences of mitotic arrest after washout of microtubule-stabilizing drugs. The initial event of relatively short treatment with microtubule stabilizers of cell cultures prone to mitotic slippage is formation of the numerous multi-nucleated cells. Restoration of normal divisions takes long time. We address the question what cells are the source of delayed recovery after drug washout.
Find out more: Suleimenov M, Bekbayev S, Ten M, Suleimenova N, Tlegenova M, Nurmagambetova A, Kauanova S and Vorobjev I (2022) Bcl-xL activity influences outcome of the mitotic arrest. Front. Pharmacol. 13:933112. doi: 10.3389/fphar.2022.933112
MEMBERS
- Ivan VorobyevProfessor and Laboratory Head
- Marina JanibekovaJunior Researcher
- Arina Zhuravel
Research Assistant
- Vadim MustyatsaResearch Assistant
EQUIPMENT
Zeiss Cell Observer Microscope
This system can be used to perform multidimensional high speed imaging of cell cultures; combination of multichannel, time lapse and Z-stack with fast acquisition, Imaging of most of the available fluorescent proteins (BFP, CFP, GFP, YFP, DsRed, mRFP, HcRed, mCherry etc.).
Applications
● Long-term observation of physiological and morphological changes in normal and cancer cells
● Evaluate and document transfection rate and transfection stability
● Identification, quantification and qualification of fluorescently labeled cells, cell structures
● Multidimensional Acquisition: time lapse series, Z-stacks, multichannel, mosaic images, multiposition imaging, optical sectioning
Manufacturer Carl Zeiss
Model Cell Observer
Public Location C4, 607
Quantity 1
We have following light sources:
● Colibri LED illumination system: LED module 365 nm, LED module 455 nm, LED module 505 nm, LED module 590 nm
● HXP 120 illumination equipment (Metal-halide lamp as additional white light source)
We have following filtersets to observe samples: DAPI, HE eGFP, HE Cy 3, Cy 5, HE CFP+YFP, HE BFP + GFP + HcRed
● Long-term observation of physiological and morphological changes in normal and cancer cells
● Evaluate and document transfection rate and transfection stability
● Identification, quantification and qualification of fluorescently labeled cells, cell structures
● Multidimensional Acquisition: time lapse series, Z-stacks, multichannel, mosaic images, multiposition imaging, optical sectioning
Manufacturer Carl Zeiss
Model Cell Observer
Public Location C4, 607
Quantity 1
We have following light sources:
● Colibri LED illumination system: LED module 365 nm, LED module 455 nm, LED module 505 nm, LED module 590 nm
● HXP 120 illumination equipment (Metal-halide lamp as additional white light source)
We have following filtersets to observe samples: DAPI, HE eGFP, HE Cy 3, Cy 5, HE CFP+YFP, HE BFP + GFP + HcRed
Heracell™ 150i & 240i CO2 Incubator, 150L & 240L
The incubator offers an optimal in vitro environment. It is characterized by cleanliness, reliability, and user-friendly operation, ensuring the protection of valuable samples. Additionally, it enhances cell growth through rapid recovery features and a convenient touchscreen interface.
SafeMate ECO 1.5 - CLASS II MICROBIOLOGICAL SAFETY CABINET
The primary purpose of the Class II Biosafety Cabinet is to manage tasks necessitating a sterile workspace. This type of biological safety cabinet is structured with several key components: an inlet to shield the operator, a ULPA/HEPA-filtered downward airflow generating an ISO Class 3 workspace to prevent sample cross-contamination, and a ULPA/HEPA-filtered exhaust to safeguard the surrounding environment.