Giant obscurin regulates migration and metastasis via RhoA-dependent cytoskeletal remodeling in pancreatic cancer.

Obscurins, encoded by the OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. Large scale omics analyses reveal that OBSCN is highly mutated across different types of cancer, exhibiting a 5-8% mutation frequency in pancreatic cancer. Yet, the functional role of OBSCN in pancreatic cancer progression and metastasis has to be delineated. We herein show that giant obscurins are highly expressed in normal pancreatic tissues, but their levels are markedly reduced in pancreatic ductal adenocarcinomas. Silencing of giant obscurins in non-tumorigenic Human Pancreatic Ductal Epithelial (HPDE) cells and obscurin-expressing Panc5.04 pancreatic cancer cells induces an elongated, spindle-like morphology and faster cell migration via cytoskeletal remodeling. Specifically, depletion of giant obscurins downregulates RhoA activity, which in turn results in reduced focal adhesion density, increased microtubule growth rate and faster actin dynamics. Although OBSCN knockdown is not sufficient to induce de novo tumorigenesis, it potentiates tumor growth in a subcutaneous implantation model and exacerbates metastasis in a hemispleen murine model of pancreatic cancer metastasis, thereby shortening survival. Collectively, these findings reveal a critical role of giant obscurins as tumor suppressors in normal pancreatic epithelium whose loss of function induces RhoA-dependent cytoskeletal remodeling, and promotes cell migration, tumor growth and metastasis.

The histone modification H3 lysine 27 tri-methylation has conserved gene regulatory roles in the triplicated genome of Brassica rapa L.

Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.

A microfluidic assay for the quantification of the metastatic propensity of breast cancer specimens.

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast cancer specimens, for the assessment of their metastatic propensity and for the rapid screening of potential antimetastatic therapeutics. On the basis of the key roles of cell motility and proliferation in cancer metastasis, the device accurately predicts the metastatic potential of breast cancer cell lines and of patient-derived xenografts. Compared with unsorted cancer cells, highly motile cells isolated by the device exhibited similar tumourigenic potential but markedly increased metastatic propensity in vivo. RNA sequencing of the highly motile cells revealed an enrichment of motility-related and survival-related genes. The approach might be developed into a companion assay for the prediction of metastasis in patients and for the selection of effective therapeutic regimens.

A Direct Podocalyxin-Dynamin-2 Interaction Regulates Cytoskeletal Dynamics to Promote Migration and Metastasis in Pancreatic Cancer Cells.

The sialoglycoprotein podocalyxin is absent in normal pancreas but is overexpressed in pancreatic cancer and is associated with poor clinical outcome. Here, we investigate the role of podocalyxin in migration and metastasis of pancreatic adenocarcinomas using SW1990 and Pa03c as cell models. Although ezrin is regarded as a cytoplasmic binding partner of podocalyxin that regulates actin polymerization via Rac1 or RhoA, we did not detect podocalyxin-ezrin association in pancreatic cancer cells. Moreover, depletion of podocalyxin did not alter actin dynamics or modulate Rac1 and RhoA activities in pancreatic cancer cells. Using mass spectrometry, bioinformatics analysis, coimmunoprecipitation, and pull-down assays, we discovered a novel, direct binding interaction between the cytoplasmic tail of podocalyxin and the large GTPase dynamin-2 at its GTPase, middle, and pleckstrin homology domains. This podocalyxin-dynamin-2 interaction regulated microtubule growth rate, which in turn modulated focal adhesion dynamics and ultimately promoted efficient pancreatic cancer cell migration via microtubule- and Src-dependent pathways. Depletion of podocalyxin in a hemispleen mouse model of pancreatic cancer diminished liver metastasis without altering primary tumor size. Collectively, these findings reveal a novel mechanism by which podocalyxin facilitates pancreatic cancer cell migration and metastasis. SIGNIFICANCE: These findings reveal that a novel interaction between podocalyxin and dynamin-2 promotes migration and metastasis of pancreatic cancer cells by regulating microtubule and focal adhesion dynamics.

Development of an optogenetic toolkit for neural circuit dissection in squirrel monkeys.

Optogenetic tools have opened a rich experimental landscape for understanding neural function and disease. Here, we present the first validation of eight optogenetic constructs driven by recombinant adeno-associated virus (AAV) vectors and a WGA-Cre based dual injection strategy for projection targeting in a widely-used New World primate model, the common squirrel monkey Saimiri sciureus. We observed opsin expression around the local injection site and in axonal projections to downstream regions, as well as transduction to thalamic neurons, resembling expression patterns observed in macaques. Optical stimulation drove strong, reliable excitatory responses in local neural populations for two depolarizing opsins in anesthetized monkeys. Finally, we observed continued, healthy opsin expression for at least one year. These data suggest that optogenetic tools can be readily applied in squirrel monkeys, an important first step in enabling precise, targeted manipulation of neural circuits in these highly trainable, cognitively sophisticated animals. In conjunction with similar approaches in macaques and marmosets, optogenetic manipulation of neural circuits in squirrel monkeys will provide functional, comparative insights into neural circuits which subserve dextrous motor control as well as other adaptive behaviors across the primate lineage. Additionally, development of these tools in squirrel monkeys, a well-established model system for several human neurological diseases, can aid in identifying novel treatment strategies.

Breeding of lilies and tulips-Interspecific hybridization and genetic background.

Lilies and tulips (Liliaceae family) are economically very important ornamental bulbous plants. Here, we summarize major breeding goals, the role of an integrated method of cut-style pollination and fertilization followed by embryo rescue and mitotic and meiotic polyploidization involved in new assortment development. Both crops have been subjected to extensive interspecific hybridization followed by selection. Additionally, spontaneous polyploidization has played a role in their evolution. In lilies, there is a tendency to replace diploids with polyploid cultivars, whereas in tulip a majority of the cultivars that exist today are still diploid except for triploid Darwin hybrid tulips. The introduction of molecular cytogenetic techniques such as genomic in situ hybridization (GISH) permitted the detailed studies of genome composition in lily and tulip interspecific hybrids and to follow the chromosome inheritance in interspecific crosses. In addition, this review presents the latest information on phylogenetic relationship in lily and tulip and recent developments in molecular mapping using different DNA molecular techniques.

Overexpression of VLA-4 in glial-restricted precursors enhances their endothelial docking and induces diapedesis in a mouse stroke model.

The loss of oligodendrocytes after stroke is one of the major causes of secondary injury. Glial-restricted progenitors (GRPs) have remylenating potential after intraparenchymal cerebral transplantation. The intraarterial (IA) injection route is an attractive gateway for global brain delivery, but, after IA infusion, naive GRPs fail to bind to the cerebral vasculature. The aim of this study was to test whether overexpression of Very Late Antigen-4 (VLA-4) increases endothelial docking and cerebral homing of GRPs in a stroke model. Mouse GRPs were co-transfected with DNA plasmids encoding VLA-4 subunits (α4, ß1). The adhesion capacity and migration were assessed using a microfluidic assay. In vivo imaging of the docking and homing of IA-infused cells was performed using two-photon microscopy in a mouse middle cerebral artery occlusion (MCAO) model. Compared to naïve GRPs, transfection of GRPs with VLA-4 resulted in >60% higher adhesion (p < 0.05) to both purified Vascular Cell Adhesion Molecule-11 (VCAM-11) and TNFα-induced endothelial VCAM-1. VLA-4+GRPs displayed a higher migration in response to a chemoattractant gradient. Following IA infusion, VLA-4+GRPs adhered to the vasculature at three-fold greater numbers than naïve GRPs. Multi-photon imaging confirmed that VLA-4 overexpression increases the efficiency of GRP docking and leads to diapedesis after IA transplantation. This strategy may be further exploited to increase the efficacy of cellular therapeutics.

E-selectin-mediated rolling facilitates pancreatic cancer cell adhesion to hyaluronic acid.

Tumor cell extravasation is a multistep process preceded by cell rolling and arrest on the vessel wall via the formation of specific receptor-ligand bonds. The strength, availability, and number of receptor-ligand bonds regulate the rate by which tumor cells tether, roll, and adhere to vascular walls. Although the mechanics of selectin-mediated rolling have been extensively studied, little is known regarding how tumor cell rolling on selectins facilitates adhesion to a distinct substrate-bound protein with different kinetic properties. By using multicomponent protein patterning and a microfluidic system, we evaluated how E-selectin-dependent rolling modulates hyaluronic acid (HA) adhesion as a function of fluid shear, contact time, and the spacing between E-selectin and HA regions patterned on the substrate. We show that tumor cells rolling on E-selectin were ∼40-fold more likely to bind to HA than nonrolling cells in shear flow. Furthermore, E-selectin-dependent rolling promotes adhesion to HA by both physically slowing cells and enabling them to position proximal to the surface, thereby increasing the on rate of adhesion. A better understanding of tumor cell adhesion under physiologic shear would lead to the development of new diagnostic assays and pave the way to clinical approaches aimed ultimately to halt metastasis.-Shea, D. J., Li, Y. W., Stebe, K. J., Konstantopoulos, K. E-selectin-mediated rolling facilitates pancreatic cancer cell adhesion to hyaluronic acid.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress.

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.

Interplay of the physical microenvironment, contact guidance, and intracellular signaling in cell decision making.

The peritumoral physical microenvironment consists of complex topographies that influence cell migration. Cell decision making, upon encountering anisotropic, physiologically relevant physical cues, has yet to be elucidated. By integrating microfabrication with cell and molecular biology techniques, we provide a quantitative and mechanistic analysis of cell decision making in a variety of well-defined physical microenvironments. We used MDA-MB-231 breast carcinoma and HT1080 fibrosarcoma as cell models. Cell decision making after lateral confinement in 2-dimensional microcontact printed lines is governed by branch width at bifurcations. Cells confined in narrow feeder microchannels prefer to enter wider branches at bifurcations. In contrast, in feeder channels that are wider than the cell body, cells elongate along one side wall of the channel and are guided by contact with the wall to the contiguous branch channel independent of its width. Knockdown of ß1-integrins or inhibition of cellular contractility suppresses contact guidance. Concurrent, but not individual, knockdown of nonmuscle myosin isoforms IIA and IIB also decreases contact guidance, which suggests the existence of a compensatory mechanism between myosin IIA and myosin IIB. Conversely, knockdown or inhibition of cell division control protein 42 homolog promotes contact guidance-mediated decision making. Taken together, the dimensionality, length scales of the physical microenvironment, and intrinsic cell signaling regulate cell decision making at intersections.-Paul, C. D., Shea, D. J., Mahoney, M. R., Chai, A., Laney, V., Hung, W.-C., Konstantopoulos, K. Interplay of the physical microenvironment, contact guidance, and intracellular signaling in cell decision making.

Distinct kinetic and mechanical properties govern mucin 16- and podocalyxin-mediated tumor cell adhesion to E- and L-selectin in shear flow.

Selectin-mediated tumor cell tethering to host cells, such as vascular endothelial cells, is a critical step in the process of cancer metastasis. We recently identified sialofucosylated mucin16 (MUC16) and podocalyxin (PODXL) as the major functional E- and L-selectin ligands expressed on the surface of metastatic pancreatic cancer cells. While the biophysics of leukocyte binding to selectins has been well studied, little is known about the mechanics of selectin-mediated adhesion pertinent to cancer metastasis. We thus sought to evaluate the critical parameters of selectin-mediated pancreatic tumor cell tethering and rolling. Using force spectroscopy, we characterized the binding interactions of MUC16 and PODXL to E- and L-selectin at the single-molecule level. To further analyze the response of these molecular interactions under physiologically relevant regimes, we used a microfluidic assay in conjunction with a mathematical model to study the biophysics of selectin-ligand binding as a function of fluid shear stress. We demonstrate that both MUC16 and PODXL-E-selectin-mediated interactions are mechanically stronger than like L-selectin interactions at the single-molecule level, and display a higher binding frequency at all contact times. The single-molecule kinetic and micromechanical properties of selectin-ligand bonds, along with the number of receptor-ligand bonds needed to initiate tethering, regulate the average velocity of ligand-coated microspheres rolling on selectin-coated surfaces in shear flow. Understanding the biophysics of selectin-ligand bonds and their responses to physiologically relevant shear stresses is vital for developing diagnostic assays and/or preventing the metastatic spread of tumor cells by interfering with selectin-mediated adhesion.