to integrate comes from in vitro studies, mainly concerning movement across two-dimensional substrata. We *Center for Biomedical Engineering and Department of Chemical Engineering nonetheless believe that much of the mechanistic understanding is relevant and useful for in vivo situations even Massachusetts Institute of Technology Cambridge, Massachusetts 02139 in three dimensions. It is likely that cells interact with their surroundings by means of the same types of recep- † Department of Cell and Structural Biology University of Illinois at Urbana-Champaign tors in vivo as in vitro, and that physical interactions of cells with their environment play important roles in Urbana, Illinois 61801 regulating function in both cases. We further attempt to suggest generalizations across Perspective a wide spectrum of migratory cell types, including amoe-Cell migration plays a central role in a wide variety of bae, leukocytes, fibroblasts, and neurons, looking for biological phenomena. In embryogenesis, cellular mibroad similarities among physical mechanisms. Obsergrations are a recurring theme in important morphogenic vations of various cells demonstrating rapid, slow, or processes ranging from gastrulation to development of negligible locomotion on particular substrata may be

Machine learning was applied for the automated derivation of causal influences in cellular signaling networks. This derivation relied on the simultaneous measurement of multiple phosphorylated protein and phospholipid components in thousands of individual primary human immune system cells. Perturbing these cells with molecular interventions drove the ordering of connections between pathway components, wherein Bayesian network computational methods automatically elucidated most of the traditionally reported signaling relationships and predicted novel interpathway network causalities, which we verified experimentally. Reconstruction of network models from physiologically relevant primary single cells might be applied to understanding native-state tissue signaling biology, complex drug actions, and dysfunctional signaling in diseased cells.

Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and β1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic 1 – 8 . For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in nonhuman primates. 52 rhesus macaques were immunized with Ad26 vectors encoding S variants or sham control and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes 9 , 10 . The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs following SARS-CoV-2 challenge. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in nonhuman primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

Recent studies have reported protective efficacy of both natural immunity 1 and vaccine-induced immunity 2 – 7 against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge in rhesus macaques. However, the importance of humoral and cellular immunity for protection against SARS-CoV-2 infection remains to be determined. Here we show that adoptive transfer of purified IgG from convalescent macaques protects naïve recipient rhesus macaques against SARS-CoV-2 challenge in a dose dependent fashion. Depletion of CD8+ T cells in convalescent animals partially abrogated the protective efficacy of natural immunity against SARS-CoV-2 re-challenge, suggesting the importance of cellular immunity in the context of waning or subprotective antibody titers. These data demonstrate that relatively low antibody titers are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may also contribute to protection if antibody responses are suboptimal. We also show that higher antibody titers are required for therapy of SARS-CoV-2 infection in macaques. These findings have important implications for the development of SARS-CoV-2 vaccines and immune-based therapeutics.

Signal transduction pathways control cellular responses to stimuli, but it is unclear how molecular information is processed as a network. We constructed a systems model of 7980 intracellular signaling events that directly links measurements to 1440 response outputs associated with apoptosis. The model accurately predicted multiple time-dependent apoptotic responses induced by a combination of the death-inducing cytokine tumor necrosis factor with the prosurvival factors epidermal growth factor and insulin. By capturing the role of unsuspected autocrine circuits activated by transforming growth factor-alpha and interleukin-1alpha, the model revealed new molecular mechanisms connecting signaling to apoptosis. The model derived two groupings of intracellular signals that constitute fundamental dimensions (molecular "basis axes") within the apoptotic signaling network. Projection along these axes captures the entire measured apoptotic network, suggesting that cell survival is determined by signaling through this canonical basis set.

Apoptosis in response to TRAIL or TNF requires the activation of initiator caspases, which then activate the effector caspases that dismantle cells and cause death. However, little is known about the dynamics and regulatory logic linking initiators and effectors. Using a combination of live-cell reporters, flow cytometry, and immunoblotting, we find that initiator caspases are active during the long and variable delay that precedes mitochondrial outer membrane permeabilization (MOMP) and effector caspase activation. When combined with a mathematical model of core apoptosis pathways, experimental perturbation of regulatory links between initiator and effector caspases reveals that XIAP and proteasome-dependent degradation of effector caspases are important in restraining activity during the pre-MOMP delay. We identify conditions in which restraint is impaired, creating a physiologically indeterminate state of partial cell death with the potential to generate genomic instability. Together, these findings provide a quantitative picture of caspase regulatory networks and their failure modes.