cellular mechanics and Force Dynamics
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Probing membrane tension dynamics via live cell tether assay
A lectin-coated bead is used to pull out a membrane tether from the cell via binding to its surface displayed glycoproteins. This physical tether allows us to directly probe in a non-invasive manner the membrane tension dynamic, e.g., ~15 pN for live HL-60 cells.
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Invincible cell membrane tethers
In a dual-trap experiments, two beads are used to pull out a pair of tethers from a live human neutrophil. These tethers are extremely flexible, with no cytoskeleton underneath, and remain sturdy under aggressive manual pulling.
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Membrane tether assay reveals cortical tension dynamics in live cells
Upon opto-genetically activated cell protrusion (actin polymerization) or contraction (actin sliding), a change in the cell membrane tension profile can be directly probed by the membrane tether assay and is distinctive to the underlying cortical dynamics.
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No tension propagation when pulling on plasma membrane alone
Given the presence of membrane-to-cortex attachments (MCA), actin cortex as the rigid component resists membrane tension propagation when external forces are applied to the membrane alone, as seen in the unperturbed tension profile of trap 2 static tether above.
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Mechanical perturbation via cortex is the key to drive tension propagation
Mechanical forces engaging only the actin cortex (i.e., endogenously via opto-driven cell protrusion/contraction; see video on the left) or both the membrane and cortex (i.e., via external aspiration shown above) effectively elicit robust membrane tension propagation in cells.
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Mitosis - a highly dynamic and mechanically intense process
During metaphase, condensed chromosomes (cyan) lined up towards the cell center by the spindle, which assembly tumbles around before it is anchored to the cell cortex and membrane, thereby defining the equator plane where the cell will eventually furrow and divide into two.
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Visualize dynamic spindle assembly in live mouse embryonic stem cells
We can use our adaptive optical tweezers instrument to monitor in real-time the progression of chromosome segregation in a mitotic mESC (DNAs labeled in green; spindle in red, top-down view) and simultaneously probe its cortical tension dynamics around the cell.
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Synchronized mitosis during embryo development
Via live cell membrane tether assay in a dual trap experiment, we aim to simultaneously monitor the cortical tension dynamics of a dividing cell and its neighbors within an embryo (image ref), thereby resolving their coordination/synchronicity during early stage embryogenesis.
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Mitosis reversed/interrupted in compromised sea star embryo
One of the dividing cells in a sea star embryo (membrane stained, shown in green) does not tolerate well the DNA stain (NucRed) and shrinks back after elongating during mitosis. We want to know how its cortical tension dynamics collapses, leading to aborted mitosis.
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Nucleus under mechanical perturbation
With the refined manipulating power of optical traps, we can use beads to apply physiological level of forces to indent or stretch nuclei (cyan) in live mESCs and study how mechanical perturbations impact chromatin organization, gene accessibility, and transcription dynamics. -
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Tether a single piece of DNA, RNA, or protein between two beads
We can use optical trap and micropipette to control a pair of micron-sized beads, which surfaces specifically bind to the two ends of a biomolecule (e.g., via biotin:streptavidin or anti-DIG:DIG interaction), thereby applying force to unfold/refold the molecule, such as MAD2.
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Resolve the mechanical forces during dynamic membrane remodeling
Our integrated optical tweezers allow us to incisively probe the ATP-dependent force generation by ESCRT complexes, which remodel the plasma membrane and sever the membrane neck (image ref; see next video on the right for details) of an outward budding vesicle.
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Membrane nanotube snapped by GUV-encapsulated ESCRT complex
A trap-controlled bead pulls out a nanotube from a vesicle (GUV) held on a micropipette. The encapsulated ESCRT complex then hydrolyzes the UV-uncaged ATPs to remodel membrane at the tube base (red box), exerting forces >60 pN to snap back the tube/bead.
Cell-Bio-Physics
Cells and biomolecules (DNA, RNA & proteins) are physical objects possessing unique mechanical properties and responding to specific mechanical cues such as tensions and forces. Their physical characteristics critically impact how they carry out basic operations in biological processes over a broad range of scales, e.g., from chromosome segregation during mitosis, nuclear mechano-transduction in cell lineage differentiation, to embryo development adapting shape and morphology. Yet, this mechanical dimension in biology has often been overlooked — partly due to limited experimental approaches available — and holds great potential to resolve aberrant phenomena unrelated to genomic mutations as found in certain cancer cell proliferation and tumorigenesis.
Recently, we succeed to study live cells force dynamics using adaptive high-resolution optical tweezers integrated with confocal fluorescence imaging module. In collaboration with the Weiner Lab at UCSF, we implemented optogenetic controls to modulate cellular activities on our force-probing/physical-manipulating platform (optical trapping plus micropipette aspiration; see videos above), which allows us to mechanically perturb the cells and simultaneously measure cellular forces such as change in membrane tension. We discovered that actin-driven protrusions/contractions in live human neutrophils generate rapid long-range membrane tension propagation across the cell, a property essential to the establishment of cell polarity during migration, and settled a long dispute in the field. This work also reveals that our membrane tether assay serves as an incisive approach to non-invasively resolve cortical tensions inside the cell, which becomes the rigorous basis to launch our studies on cortical dynamics during mitosis. By customizing our microfluidic setup, we expand the variety of cell types amenable to our system, ranging from mouse embryonic stem cells (mESCs) to an actively dividing sea star embryo (collaboration with the Barone Lab at Stanford).
How does the cell ‘physically’ divide? Detailed mechanical crosstalk between the membrane, cortex, and microtubule network allows the cell to coordinate temporarily and spatially its precise changes in morphology during mitosis and promote accurate segregation of genomic material. I aim to resolve the orchestration of cortical tension between the bipolar ends and the equator in symmetric cell division, interrogate how cortical tension dynamics impact spindle positioning and determine the division plane for cytokinetic furrow ingression, and ask whether a tension/force imbalance along the spindle may lead to chromosome misalignment. — Carly
Membrane tether assays
to probe cortical tension
dynamics at multiple
locations around mESCs
How does the mitotic checkpoint proteins, MAD2, couple their metamorphic fold-switching dynamics to the precise orchestration of synchronized events during cell division? Its structural dynamics not only safeguards chromosome segregation, but also exceeds the predicting power of Alpha-Fold, and challenges the existing notion of ‘one-sequence-one-fold.’ In this project, we use single-molecule optical tweezers assay to resolve the dynamic folding trajectories for MAD2 transitions between its two functional states, which structural insight will guide us to unravel in live cells how MAD2 fold-switching flux oscillates—in time and space—accordingly with phases throughout mitosis. — Chuofan
Follow MAD2 fold switching
between open and closed forms
via optical tweezers assay
During early stage development, mitosis appears nearly concerted among cells within an embryo. How do the cortical tension dynamics evolve and coordinate between neighboring cells? Via live cell membrane tether assay in a dual trap experiment, we aim to simultaneously monitor the membrane tension profiles of cell pairs around a sea star embryo, thereby resolving their correlation and mechanical attribution to synchronicity in cell cycle. — June (joint with Barone Lab)
Synchronized furrow ingression between two neighboring mitotic cells (confocal z-slice recored at
the equator plane in real-
time; 2-to-4 cell stage).
As the mitotic cell elongates (top one; 4-to-8 cell stage), membrane tension increases near the polar end.
Ultimately, we want to study cellular mechanics in situ. Hence, we are also developing new genetically encoded force sensors and tension gauges, and constructing novel imaging platform ad hoc to directly visualize these hidden physical parameters present inside and exerted by the cells. For instance, the amount of forces transduced across each spindle fiber during mitosis, or a global map of membrane tension distribution as the entire embryo divides.
We will use optical tweezers to calibrate each molecular probe and identify constructs with proper dynamic ranges for probing cellular forces and tensions. The precise magnitude and dynamic coordination of these physical parameters are key to discern how cells interpret and integrate mechanical cues both from within and among interfacing neighbors, which is the core of our lab’s research. — Alice
Given the presence of physical linkages (e.g., LINC complexes) between the cell membrane, cortex, and nucleus, could external physical stimuli—such as the intercellular mechanical crosstalk between neighboring cells within a developing embryo—have an intracellular impact by altering chromatin organization, gene accessibility, transcription dynamics, thereby giving cues for cell fate/differentiation? With the refined manipulating power of our adaptive optical tweezers instrument, we can apply physiological level of forces to indent or stretch nuclei in live mESCs and simultaneously monitor with confocal fluorescence imaging to study how mechanical perturbations impact chromatin organization, gene accessibility/expression, and transcription dynamics.
Mechanically indent and stretch the nuclei inside live cells with a micron-sized bead controlled by the laser trap. The extent of nucleus shape change produced by these external forces is similar to that when being compressed by a neighboring mitotic cell that elongates and expands in size (see left set of images).
