Robert Wysolmerski

Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study.

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation.

Dorsal Root Ganglion Neurons React to Semaphorin 3A Application through a Biphasic Response that Requires Multiple Myosin II Isoforms

Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction.

Nonmuscle myosin II is a critical regulator of clathrin-mediated endocytosis.

Variable requirements for actin during clathrin‐mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) stimulation of embryonic mouse sensory axon outgrowth is MII dependent. NGF regulates two actomyosin processes: transverse actin bundling and peripheral retrograde (radial) network actin flow. These two processes oppose microtubule advance and differentially involve MIIA and MIIB, respectively.

Bone marrow osteoblast vulnerability to chemotherapy

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy‐induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post‐transplantation.

Non-Muscle Myosin II Isoforms Have Different Functions in Matrix Rearrangement by MDA-MB-231 Cells.

The role of a stiffening extra-cellular matrix (ECM) in cancer progression is documented but poorly understood. Here we use a conditioning protocol to test the role of nonmuscle myosin II isoforms in cell mediated ECM arrangement using collagen constructs seeded with breast cancer cells expressing shRNA targeted to either the IIA or IIB heavy chain isoform. While there are several methods available to measure changes in the biophysical characteristics of the ECM, we wanted to use a method which allows for the measurement of global stiffness changes as well as a dynamic response from the sample over time. The conditioning protocol used allows the direct measurement of ECM stiffness. Using various treatments, it is possible to determine the contribution of various construct and cellular components to the overall construct stiffness. Using this assay, we show that both the IIA and IIB isoforms are necessary for efficient matrix remodeling by MDA-MB-231 breast cancer cells, as loss of either isoform changes the stiffness of the collagen constructs as measured using our conditioning protocol. Constructs containing only collagen had an elastic modulus of 0.40 Pascals (Pa), parental MDA-MB-231 constructs had an elastic modulus of 9.22 Pa, while IIA and IIB KD constructs had moduli of 3.42 and 7.20 Pa, respectively. We also calculated the cell and matrix contributions to the overall sample elastic modulus. Loss of either myosin isoform resulted in decreased cell stiffness, as well as a decrease in the stiffness of the cell-altered collagen matrices. While the total construct modulus for the IIB KD cells was lower than that of the parental cells, the IIB KD cell-altered matrices actually had a higher elastic modulus than the parental cell-altered matrices (4.73 versus 4.38 Pa). These results indicate that the IIA and IIB heavy chains play distinct and non-redundant roles in matrix remodeling.

Three-dimensional nonlinear microspectroscopy and imaging of soft condensed matter.

We report on the realization of a sensitive microspectroscopy and imaging approach based on a three-color femtosecond coherent anti-Stokes Raman scattering (CARS) technique with high spectral, time, and spatial resolution. Independently tunable, high-repetition rate optical parametric oscillators were used to attain a dynamic range of 5 orders of magnitude for time-domain CARS signal. The attained sensitivity permitted tracing the decay of weak and structurally complex Raman active modes in soft condensed matter. Application of this approach to imaging of the biological specimen shows a great potential in quantitative characterization of live biological media with an ability to access inter- and intra-molecular interactions.

Abstract A66: NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling

The adhesion scaffolding protein NEDD9 was identified as potential pro-metastatic gene in several cancers. The molecular mechanisms of NEDD9-driven metastasis are still unknown. In this study, we show that expression of NEDD9 positively correlates with the invasive stage of breast cancer. We show that NEDD9 localizes to invadopodia and endosomes. Notably, NEDD9 depleted cells have increased levels of inactive surface receptors due to increase in fast recycling of Rab4 and Rab5 positive vesicles. Mechanistically, we found that NEDD9 binds to and scaffolds the Arf6 specific GAP - ASAP3, decreasing Arf6 activity. Thus, depletion of NEDD9 leads to activation of Arf6. Inhibition of Arf6 or re-expression of NEDD9 in shNEDD9 cells was sufficient to restore recycling rates, decrease the number of Rab4 and 5 positive vesicles and the invasive properties of tumor cells. Thus, in this work, we uncover the mechanistic basis of NEDD9-driven invasion and identify a new role for NEDD9 in Arf6-dependant endocytosis. Citation Format: Elena N. Pugacheva, Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Mark Culp, Robert Wysolmerski, Scott Weed, Alexey Ivanov. NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A66.

Abstract 3782: NEDD9 promotes cell invasion through modulation of Arf6-dependent endocytic recycling.

The adaptor protein, NEDD9, is an established pro-metastatic trigger in several cancers but the molecular mechanisms behind NEDD9-driven invasion remain unclear. Here we show that expression of NEDD9 protein tightly correlates with the transition of breast cancer to invasive stages. Overexpression of NEDD9 is critical for the invasion of cancer cells but depletion of NEDD9 is sufficient to block invasion. It is also well established that invadopodia are important for cell invasion. We demonstrate that NEDD9 localizes to invadopodia and its depletion leads to a deficiency in matrix degradation. NEDD9 depletion was also accompanied by an increase in surface levels of inactive proteases (MT1-MMP) and adhesion receptors (integrins). In addition, depletion of NEDD9 induced an increase in recycling and early endosomes via activation of Arf6. NEDD9 binds to the Arf6 specific GAP, ASAP3, which explains NEDD9 modulation of Arf6 activity. Inhibition of Arf6 or re-expression of NEDD9 rescue in NEDD9-deficient cells is sufficient to restore proper recycling rates and invasive properties of breast cancer cells. These results reveal the mechanism behind NEDD9-driven migration and tumor invasion. Citation Format: Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Anuradha Rajulapati, Mark Culp, Robert Wysolmerski, Alexey Ivanov, Scott Weed, Elena Pugacheva. NEDD9 promotes cell invasion through modulation of Arf6-dependent endocytic recycling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2013-3782

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

Summary Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.