Unite to divide - how models and biological experimentation have come together to reveal mechanisms of cytokinesis.

Cytokinesis is the fundamental and ancient cellular process by which one cell physically divides into two. Cytokinesis in animal and fungal cells is achieved by contraction of an actomyosin cytoskeletal ring assembled in the cell cortex, typically at the cell equator. Cytokinesis is essential for the development of fertilized eggs into multicellular organisms and for homeostatic replenishment of cells. Correct execution of cytokinesis is also necessary for genome stability and the evasion of diseases including cancer. Cytokinesis has fascinated scientists for well over a century, but its speed and dynamics make experiments challenging to perform and interpret. The presence of redundant mechanisms is also a challenge to understand cytokinesis, leaving many fundamental questions unresolved. For example, how does a disordered cytoskeletal network transform into a coherent ring? What are the long-distance effects of localized contractility? Here, we provide a general introduction to 'modeling for biologists', and review how agent-based modeling and continuum mechanics modeling have helped to address these questions.

A free-boundary model of a motile cell explains turning behavior.

To understand shapes and movements of cells undergoing lamellipodial motility, we systematically explore minimal free-boundary models of actin-myosin contractility consisting of the force-balance and myosin transport equations. The models account for isotropic contraction proportional to myosin density, viscous stresses in the actin network, and constant-strength viscous-like adhesion. The contraction generates a spatially graded centripetal actin flow, which in turn reinforces the contraction via myosin redistribution and causes retraction of the lamellipodial boundary. Actin protrusion at the boundary counters the retraction, and the balance of the protrusion and retraction shapes the lamellipodium. The model analysis shows that initiation of motility critically depends on three dimensionless parameter combinations, which represent myosin-dependent contractility, a characteristic viscosity-adhesion length, and a rate of actin protrusion. When the contractility is sufficiently strong, cells break symmetry and move steadily along either straight or circular trajectories, and the motile behavior is sensitive to conditions at the cell boundary. Scanning of a model parameter space shows that the contractile mechanism of motility supports robust cell turning in conditions where short viscosity-adhesion lengths and fast protrusion cause an accumulation of myosin in a small region at the cell rear, destabilizing the axial symmetry of a moving cell.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery.

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

In relapsed or refractory diffuse large B-cell lymphoma, CD19 expression by immunohistochemistry alone is not a predictor of response to loncastuximab tesirine.

CD19-targeting treatments have shown promise in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Loncastuximab tesirine (loncastuximab tesirine-lpyl [Lonca]) is a CD19-targeting antibody-drug conjugate indicated for R/R DLBCL after at least two systemic treatments. CD19 expression was evaluated in patients receiving Lonca in the LOTIS-2 clinical trial with available tissue samples obtained after last systemic therapy/before Lonca treatment. Lonca cytotoxicity was evaluated in a panel of six lymphoma cell lines with various CD19 expression levels. Quantitative systems pharmacology (QSP) modelling was used to predict Lonca responses. Lonca responses were seen in patients across all CD19 expression levels, including patients with low/no detectable CD19 expression and H-scores at baseline. Similarly, Lonca induced cytotoxicity in cell lines with different levels of CD19 expression, including one with very low expression. QSP modelling predicted that CD19 expression by immunohistochemistry alone does not predict Lonca response, whereas inclusion of CD19 surface density improved response prediction. Virtual patients responded to Lonca with estimated CD19 as low as 1000 molecules/cell of CD19, normally below the immunohistochemistry detection level. We found Lonca is an effective treatment for R/R DLBCL regardless of CD19 expression by immunohistochemistry. These results provide the basis for future studies addressing CD19-targeted agent sequencing.

Using quantitative systems pharmacology modeling to optimize combination therapy of anti-PD-L1 checkpoint inhibitor and T cell engager.

Although immune checkpoint blockade therapies have shown evidence of clinical effectiveness in many types of cancer, the outcome of clinical trials shows that very few patients with colorectal cancer benefit from treatments with checkpoint inhibitors. Bispecific T cell engagers (TCEs) are gaining popularity because they can improve patients' immunological responses by promoting T cell activation. The possibility of combining TCEs with checkpoint inhibitors to increase tumor response and patient survival has been highlighted by preclinical and clinical outcomes. However, identifying predictive biomarkers and optimal dose regimens for individual patients to benefit from combination therapy remains one of the main challenges. In this article, we describe a modular quantitative systems pharmacology (QSP) platform for immuno-oncology that includes specific processes of immune-cancer cell interactions and was created based on published data on colorectal cancer. We generated a virtual patient cohort with the model to conduct in silico virtual clinical trials for combination therapy of a PD-L1 checkpoint inhibitor (atezolizumab) and a bispecific T cell engager (cibisatamab). Using the model calibrated against the clinical trials, we conducted several virtual clinical trials to compare various doses and schedules of administration for two drugs with the goal of therapy optimization. Moreover, we quantified the score of drug synergy for these two drugs to further study the role of the combination therapy.

In vitro experiments and kinetic models of Arabidopsis pollen hydration mechanics show that MSL8 is not a simple tension-gated osmoregulator.

Pollen, a neighbor-less cell containing the male gametes, undergoes mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously shown that the pollen-specific mechanosensitive ion channel MscS-like (MSL)8 is essential for pollen survival during hydration and proposed that it functions as a tension-gated osmoregulator. Here, we test this hypothesis with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell, and then they stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model, wherein MSL8 acts as a simple-tension-gated osmoregulator, does not replicate this behavior. A better fit was obtained from variations of the model, wherein MSL8 inactivates independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the cell wall strengthening model best simulated experimental responses. Finally, the expression of a nonconducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that contrary to our hypothesis and to the current understanding of MS ion channel function in bacteria, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular scale model system and illustrate how mathematical models can correct intuitive hypotheses.

A model of actin-driven endocytosis explains differences of endocytic motility in budding and fission yeast.

A comparative study (Sun et al., 2019) showed that the abundance of proteins at sites of endocytosis in fission and budding yeast is more similar in the two species than previously thought, yet membrane invaginations in fission yeast elongate twofold faster and are nearly twice as long as in budding yeast. Here we use a three-dimensional model of a motile endocytic invagination (Nickaeen et al., 2019) to investigate factors affecting elongation of the invaginations. We found that differences in turgor pressure in the two yeast species can largely explain the paradoxical differences observed experimentally in endocytic motility.

Actin assembly produces sufficient forces for endocytosis in yeast.

We formulated a spatially resolved model to estimate forces exerted by a polymerizing actin meshwork on an invagination of the plasma membrane during endocytosis in yeast cells. The model, which approximates the actin meshwork as a visco-active gel exerting forces on a rigid spherocylinder representing the endocytic invagination, is tightly constrained by experimental data. Simulations of the model produce forces that can overcome resistance of turgor pressure in yeast cells. Strong forces emerge due to the high density of polymerized actin in the vicinity of the invagination and because of entanglement of the meshwork due to its dendritic structure and cross-linking. The model predicts forces orthogonal to the invagination that are consistent with formation of a flask shape, which would diminish the net force due to turgor pressure. Simulations of the model with either two rings of nucleation-promoting factors (NPFs) as in fission yeast or a single ring of NPFs as in budding yeast produce enough force to elongate the invagination against the turgor pressure.