Biology subjects

Savich, Y.

Publications and source records attributed to Savich, Y..

4 recordsLinked to original sources

bioRxiv2025

Filopodia numbers impact chemotactic migration speed

Migrating cells sense and respond to external chemical and physical cues, enabling them to efficiently reach their destinations. Filopodia are slender actin-filled membrane protrusions implicated in interacting with the extracellular environment in many contexts, such as neuronal growth cone guidance and the capture of prey by immune cells and unicellular organisms. The role of filopodia in chemotactic guidance in fast-moving amoeboid cells has not been well-studied. The social amoeba Dictyostelium relies on chemotaxis for development and finding food, making it an excellent system for investigating the role of filopodia in amoeboid chemotaxis. Stimulation of amoebae with the chemoattractant cAMP activates a transient increase of filopodia formation by recruiting the filopodial myosin DdMyo7 to the cell cortex. Filopodia formation is biased towards the source of chemoattractant, yet myo7 null cells that lack filopodia exhibit normal directional migration. However, cells either lacking filopodia or having increased numbers of filopodia move more slowly than those with wildtype numbers of filopodia. Thus, while filopodia are dispensable for detection of chemical gradients by amoeboid cells, changes in filopodia number can impact their migration speed possibly due to altering cell-substrate adhesion. In briefFilopodia formation in chemotactic amoeboid cells is stimulated by chemoattractant and biased towards the gradient source. Lack of filopodia does not impair directed migration but rather reduces cell speed. Changes in filopodia number correlate with the speed of chemotactic cells suggesting a role for these extension in tuning adhesion for optimal migration. HighlightsO_LIFilopodia formation in chemotactic amoeboid cells is biased towards the source of the gradient. C_LIO_LIChemotactic Dictyostelium amoebae lacking filopodia migrate with directional persistence. C_LIO_LIMigration speed is altered by filopodia number. C_LI

cell biology↗

bioRxiv2025

A protein-DNA surface hydrogel mechanically protects the cell nucleus

The nuclear envelope protects the genome from mechanical stress during processes such as migration, division, and compression1-6, but how it buffers forces at the scale of DNA remains unclear. Here, we utilize optical tweezers to show that a multivalent protein-DNA co-condensate containing the nuclear envelope protein LEM27,8 and the DNA-binding protein BAF 9,10 shield DNA beyond its melting point at 65 pN11. Under load, their collective assembly induces an unconventional DNA stiffening effect that provides mechanical reinforcement, dependent on the intrinsically disordered region (IDR) of LEM2. At the nuclear surface, these components form an elastic surface hydrogel in which LEM2 IDR-IDR interactions contract the surface hydrogel relative to its relaxed state, introducing a pre-stress in the lamin network. Inside cells, this surface hydrogel model can recapitulate elastic properties of the nuclear envelope measured via AFM indentation experiments as well as nuclear morphology, using parameters obtained at the molecular scale by use of optical tweezers. Disruption of the surface hydrogel increases DNA damage and micronuclei formation during nuclear deformation. These findings reveal a load-bearing, mesoscale surface hydrogel that reinforces the nucleus and expands the functional repertoire of biomolecular condensates to include DNA protection under mechanical stress.

biophysics↗

bioRxiv2024

Active Loop Extrusion guides DNA-Protein Condensation

The spatial organization of DNA involves DNA loop extrusion and the formation of protein-DNA condensates. While the significance of each process is increasingly recognized, their interplay remains unexplored. Using molecular dynamics simulation and theory we investigate this interplay. Our findings reveal that loop extrusion can enhance the dynamics of condensation and promotes coalescence and ripening of condensates. Further, the DNA loop enables condensate formation under DNA tension and position condensates. The concurrent presence of loop extrusion and condensate formation results in the formation of distinct domains similar to TADs, an outcome not achieved by either process alone.

biophysics↗

bioRxiv2019

Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

We have measured the orientation of the myosin light chain domain (lever arm) elements in demembranated muscle fibers by electron paramagnetic resonance (EPR), using a bifunctional spin label (BSL), with angular resolution of 4 degrees. Despite advances in X-ray crystallography and cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain under ambient conditions in a muscle fiber. Two cysteines, 4 residues apart, were engineered on two -helices in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. One labeled helix (helix E) is adjacent to the myosin lever arm, the other helix (helix B) is located farther apart from the motor domain beyond the "hinge" of the myosin. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that determined angular distributions of BSL with high resolution, which enabled the accurate determination of helix orientation of individual structural elements with respect to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibited both ordered ({sigma} ~ 9-11 degrees) and disordered ({sigma} > 38 degrees) populations. We used these angles to determine the orientation of the myosin lever arm, concluding that the oriented population has lever arms that are perpendicular to the muscle fiber axis. This orientation is ~33 degrees different than predicted from a standard "lever arm down" model based on cryo-EM of actin decorated with isolated myosin heads, but it is compatible with fluorescence polarization and EM data obtained from muscle fibers. The addition of ATP, in the absence of Ca2+, shifted the orientation to a much more disordered distribution. SummaryWe used electron paramagnetic resonance to determine the orientation of elements within the myosin regulatory light chain in skinned skeletal muscle fibers. A bifunctional spin label provided sufficient resolution to detect an ordered population of lever arms perpendicular to actin.

biophysics↗