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Purpose: Glioblastoma (GBM) is an aggressive malignant brain tumor with 2 year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127–4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026–1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411–2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583–1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models. Methods: Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Results: We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells. Conclusions: Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch. [ABSTRACT FROM AUTHOR]

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis. Mechanical forces drive critical cellular processes, but methods to study such cellular forces are typically low-throughput. Here the authors present a method using "Rupture And Deliver" Tension Gauge Tethers, where flow cytometry or sequencing can be used to record the mechanical history of thousands of individual cells. [ABSTRACT FROM AUTHOR]

Microtubule "dynamic instability," the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the β subunit of the αβ-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~ 4 kBT) and an outward bending preference (~ 1.5 kBT) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment. [ABSTRACT FROM AUTHOR]

Understanding how cells migrate in fibrous environments is important in wound healing, immune function, and cancer progression. A key question is how fiber orientation and network geometry influence cell movement. Here we describe a quantitative, modeling-based approach toward identifying the mechanisms by which cellsmigrate in fibrous geometries having well controlled orientation. Specifically,U251 glioblastoma cells were seeded onto non-electrospinning Spinneret based tunable engineering parameters fiber substrates that consist of networks of suspended 400 nm diameter nanofibers. Cells were classified based on the local fiber geometry and cell migration dynamics observed by lightmicroscopy. Cells were found in three distinct geometries: adhering two a single fiber, adhering to two parallel fibers, and adhering to a network of orthogonal fibers. Cells adhering to a single fiber or two parallel fibers can onlymove in one dimension along the fiber axis,whereas cells on a network of orthogonal fibers can move in two dimensions. We found that cellsmove faster andmore persistently in 1Dgeometries than in 2D, with cell migration being faster on parallel fibers than on single fibers. To explain these behaviors mechanistically, we simulated cellmigration in the three different geometries using a motor-clutch based model for cell traction forces. Using nearly identical parameter sets for each of the three cases,we found that the simulated cells naturally replicated the reduced migration in 2Drelative to 1Dgeometries. In addition, themodestly faster 1D migration on parallel fibers relative to single fiberswas captured using a correspondingly modest increase in the number of clutches to reflect increased surface area of adhesion on parallel fibers. Overall, the integrated modeling and experimental analysis shows that cellmigration in response to varying fibrous geometries can be explained by a simple mechanical readout of geometry via a motor-clutch mechanism. [ABSTRACT FROM AUTHOR]

Introduction: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment 'congression' at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. Methods: RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. Results: RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. Conclusions: This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis. [ABSTRACT FROM AUTHOR]

Microenvironmental mechanics play an important role in determining the morphology, traction, migration, proliferation, and differentiation of cells. A stochastic motor-clutch model has been proposed to describe this stiffness sensitivity. In this work, we present a master equation-based ordinary differential equation (ODE) description of the motor-clutch model, from which we derive an analytical expression to for a cell’s optimum stiffness (i.e., the stiffness at which the traction force is maximal). This analytical expression provides insight into the requirements for stiffness sensing by establishing fundamental relationships between the key controlling cell-specific parameters. We find that the fundamental controlling parameters are the numbers of motors and clutches (constrained to be nearly equal), and the time scale of the on–off kinetics of the clutches (constrained to favor clutch binding over clutch unbinding). Both the ODE solution and the analytical expression show good agreement with Monte Carlo motor-clutch output, and reduce computation time by several orders of magnitude, which potentially enables long time scale behaviors (hours–days) to be studied computationally in an efficient manner. The ODE solution and the analytical expression may be incorporated into larger scale models of cellular behavior to bridge the gap from molecular time scales to cellular and tissue time scales. [ABSTRACT FROM AUTHOR]

The article presents the professional history of University of Michigan's Department of Biomedical Engineering professor Alan J. Hunt. The article informs that Hunt did his doctoral studies at University of Washington with another student Jon Howard. The article also informs that he undertook his postdoctoral studies at University of Colorado and faculty research at University of Michigan. The article further mentions that Hunt died on October 28, 2012.

Microtubules (MTs) are central to fundamental cellular processes including mitosis, polarization, and axon extension. A key issue is to understand how MT-associated proteins and therapeutic drugs, such as the anticancer drug paclitaxel, control MT self-assembly. To facilitate this research, it would be helpful to have automated methods that track the tip of dynamically assembling MTs as observed via fluorescence microscopy. Through a combination of digital fluorescence imaging with MT modeling, model-convolution, and automated image analysis of live and fixed MTs, we developed a method for MT tip tracking that includes estimation of the measurement error. We found that the typical single-frame tip tracking accuracy of GFP-tubulin labeled MTs in living LLC-PK1α cells imaged with a standard widefield epifluorescence digital microscope system is ~36 nm, the equivalent of ~4.5 tubulin dimer layers. However, if the MT tips are blunt, the tip tracking accuracy can be as accurate as ~15 nm (~2 dimer layers). By fitting a Gaussian survival function to the MT tip intensity profiles, we also established that MTs within living cells are not all blunt, but instead exhibit highly variable tapered tip structures with a protofilament length standard deviation of ~180 nm. More generally, the tip tracking method can be extended to track the tips of any individual fluorescently labeled filament, and can estimate filament tip structures both in vivo and in vitro with single-frame accuracy on the nanoscale. [ABSTRACT FROM AUTHOR]

The original article has been corrected Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Correction to: Annals of Biomedical Engineering (2021) https://doi.org/10.1007/s10439-020-027... The Supplementary Information in the article has been updated. [Extracted from the article]

Kinetochores maintain a mechanical grip on disassembling microtubule plus ends, possibly through a 16-member Dam1 ring that acts as a sliding clamp. It turns out, however, that a ring is not required for maintaining grip: individual Dam1 complexes in vitro can diffuse on the microtubule lattice and track shortening microtubule tips. [ABSTRACT FROM AUTHOR]

Cell migration, which is central to many biological processes including wound healing and cancer progression, is sensitive to environmental stiffness, and many cell types exhibit a stiffness optimum, at which migration is maximal. Here we present a cell migration simulator that predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction is verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ∼1 kPa) and U251 glioma cells (optimum ∼100 kPa). In addition, the model predicts, and experiments confirm, that the stiffness optimum of U251 glioma cell migration, morphology and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype. [ABSTRACT FROM AUTHOR]

An introduction to the journal is presented in which the guest editors mention that the special issue is dedicated to the professional history of Professor Alan J. Hunt who died in October 2012 of a rare for of appendix cancer.