The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.

Expression of profilin-1 (Pfn1) is downregulated in breast cancer cells, the functional significance of which is yet to be understood. To address this question, in this study we evaluated how perturbing Pfn1 affects motility and invasion of breast cancer cells. We show that loss of Pfn1 expression leads to enhanced motility and matrigel invasiveness of MDA-MB-231 breast cancer cells. Interestingly, silencing Pfn1 expression is associated with downregulation of both cell -cell and cell -matrix adhesions with concomitant increase in motility and dramatic scattering of normal human mammary epithelial cells. Thus, these data for the first time suggest that loss of Pfn1 expression may have significance in breast cancer progression. Consistent with these findings, even a moderate overexpression of Pfn1 induces actin stress-fibres, upregulates focal adhesion, and dramatically inhibits motility and matrigel invasiveness of MDA-MB-231 cells. Using mutants of Pfn1 that are defective in binding to either actin or proline-rich ligands, we further show that overexpressed Pfn1 must have a functional actin-binding site to suppress cell motility. Finally, animal experiments reveal that overexpression of Pfn1 suppresses orthotopic tumorigenicity and micro-metastasis of MDA-MB-231 cells in nude mice. These data imply that perturbing Pfn1 could be a good molecular strategy to limit the aggressiveness of breast cancer cells.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP-alpha-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP-FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP-FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP-alpha-actinin binding to the cytoplasmic domain of the beta(1) integrin subunit, but not to actin. Thus, alpha-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in alpha-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.

Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P 2 at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P 2 -binding protein] and Ena/ VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.T he ubiquitously expressed cytoskeleton-modulating protein profilin1 influences multiple processes involved in cell motility, making it a challenge to elucidate the exact molecular mechanism that controls migration. At least one major function of profilin1 is to regulate actin polymerization. Profilin1 regenerates actin monomers from disassembling filament networks by facilitating the exchange of ATP for ADP on G-actin. By further inhibiting spontaneous nucleation of G-actin, profilin1 causes an accumulation of profilin1/ATP-G-actin pool available for polymerization. Because profilin1 also has an affinity for poly-Lproline sequences, it binds to almost all major actin nucleating and F-actin elongating proteins that contain proline-rich domains [e.g., N-WASP (neuronal Wiskott-Aldrich syndrome protein), Ena (enabled)/VASP (vasodilator stimulated phosphoprotein), and formins], and this allows profilin1-mediated recruitment of ATP-G-actin to these proteins, enhancing actin polymerization (1, 2). In addition, profilin1 binds to plasma membrane presumably through its interactions with various phosphoinositides (3). Profilin1 binds to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P 2 ], and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5) P 3 ]), at least in vitro (4). Based on PI(4,5)P 2 binding, it has been proposed that the phosphoinositide binding site of profilin1 overlaps with its actin-binding site (5), and to some extent spans a second region neighboring the polyproline binding site (6). This has prompted speculation that the major role of phosphoinositide binding of profilin1 would be to inhibit its interaction with act...

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A three-dimensional numerical model is proposed to simulate the dynamic motion of red blood cells ͑RBCs͒ in simple shear flow. The RBCs are approximated by ghost cells consisting of Newtonian liquid drops enclosed by Skalak membranes which take into account the membrane shear elasticity and the membrane area incompressibility. The RBCs have an initially biconcave discoid resting shape, and the internal liquid is assumed to have the same physical properties as the matrix fluid. The simulation is based on a hybrid method, in which the immersed boundary concept is introduced into the framework of the lattice Boltzmann method, and a finite element model is incorporated to obtain the forces acting on the nodes of the cell membrane which is discretized into flat triangular elements. The dynamic motion of RBCs is investigated in simple shear flow under a broad range of shear rates. At large shear rates, the cells are found to carry out a swinging motion, in which periodic inclination oscillation and shape deformation superimpose on the membrane tank treading motion. With the shear rate decreasing, the swinging amplitude of the cell increases, and finally triggers a transition to tumbling motion. This is the first direct numerical simulation that predicts both the swinging motion of the RBCs and the shear rate induced transition, which have been observed in a recent experiment. It is also found that as the mode changes from swinging to tumbling, the apparent viscosity of the suspension increases monotonically.

We previously showed that silencing profilin-1 (Pfn1) expression increases breast cancer cell motility, but the underlying mechanisms have not been explored. Herein, we demonstrate that loss of Pfn1 expression leads to slower but more stable lamellipodial protrusion thereby enhancing the net protrusion rate and the overall motility of MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells showed dramatic enrichment of VASP at their leading edge when Pfn1 expression was downregulated and this observation was also reproducible in other cell types including human mammary epithelial cells and vascular endothelial cells. We further demonstrate that Pfn1 downregulation results in a hyper-motile phenotype of MDA-MB-231 cells in an Ena/ VASP-dependent mechanism. Pfn1-depleted cells display a strong colocalization of VASP with lamellipodin (Lpd-a PI(3,4)P 2 -binding protein that has been previously implicated in lamellipodial targeting of Ena/VASP) at the leading edge. Finally, inhibition of PI3-kinase (important for generation of PI(3,4)P 2 ) delocalizes VASP from the leading edge. This observation is consistent with a possible involvement of Lpd in enhanced membrane recruitment of VASP that results from loss of Pfn1 expression. Our findings for the first time highlight a possible mechanism of how reduced expression of a pro-migratory molecule like Pfn1 could actually promote motility of breast cancer cells.Disruption of the actin cytoskeleton is a feature of malignant cells that correlates with dysregulated expression of various actin-binding proteins (ABPs) (Wang et al., 1996;Clark et al., 2000). The malignant phenotype of tumor cells often can be reversed by experimental restoration of ABP expression, suggesting misregulation of ABPs could contribute directly to malignancy (Tanaka et al., 1995;Nikolopoulos et al., 2000). Profilin-1 (Pfn1), a ubiquitously expressed G-actin binding protein, is significantly downregulated in various types of adenocarcinoma (breast, pancreatic, hepatic) (Janke et al., 2000;Gronborg et al., 2006;Wu et al., 2006). This raises a fundamental question as to whether loss of Pfn1 expression contributes to malignant progression of tumor cells. We found that upon downregulation of Pfn1, normal human mammary epithelial cells (HMEC) exhibit disruption of cell-cell adhesion (a hallmark change that facilitates epithelial cell dissemination and migration during cancer progression) (Zou et al., 2007). Furthermore, * Correspondence to: Partha Roy, Department of Bioengineering, University of Pittsburgh, 306 Center for Bioengineering, 300 Technology Drive, Pittsburgh, PA 15219. par19@pitt.edu. Additional Supporting Information may be found in the online version of this article. NIH Public AccessAuthor Manuscript J Cell Physiol. Author manuscript; available in PMC 2010 November 24. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript silencing Pfn1 expression leads to increased motility and invasiveness of breast cancer cell lines and conversely, overexpression of Pfn...

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