Biomedical subjects

Louis Hodgson

Publications and source records attributed to Louis Hodgson.

10 recordsLinked to original sources

PubMed2006

Spatiotemporal dynamics of RhoA activity in migrating cells.

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body. However, there is evidence that RhoA also regulates membrane protrusion. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.

3T3 Cells↗

PubMed2006

Functional proteometrics for cell migration.

BACKGROUND: Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation. METHODS: We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. RESULTS: The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity. CONCLUSIONS: These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.

Animals↗

PubMed2006

Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.

This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.

Biosensing Techniques↗

PubMed2006

Digital autofocus methods for automated microscopy.

Automatic focusing of microscope images is an essential part of modern high-throughput microscopy. This chapter describes implementation of a robust autofocus system appropriate for using either air or oil immersion objectives in robotic imaging. Both hardware and software algorithms are described, and caveats of using viscous immersion media with multifield scanning are detailed.

Algorithms↗

PubMed2005

Involvement of phospholipase C signaling in melanoma cell-induced endothelial junction disassembly.

In this study, we report a phospholipase C (PLC)-mediated mechanism for the redistribution of interendothelial adherens junctions in response to melanoma cell contacts with the endothelium. We demonstrated that contact of melanoma cells to human umbilical vein endothelial cells (HUVEC) triggered rapid endothelial [Ca2+]i response through PLC-IP3 pathway. In addition, alternation of endothelial adherens junctions following contact of melanoma cells was evidenced by the changes in immunological staining patterns of vascular endothelial (VE)-cadherin. A PLC inhibitor, U73122 was shown to significantly diminish [Ca2+]i response and reduce the occurrence of melanoma cell-induced VE-cadherin reorganization. Moreover, inhibition of PLC attenuated melanoma cell transendothelial migration. However, melanoma cell-associated VE-cadherin breakdown was not sensitive to Ly294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), whereas inhibition of PI3K resulted in a reduction of melanoma cell transmigration. Taken together, our findings implicate that by inducing the PLC-Ca2+ signaling pathway, melanoma cells disrupt EC junctions to breach the endothelium and promote transvascular homing of tumor cells.

Antigens, CD↗

PubMed2004

Activation of endogenous Cdc42 visualized in living cells.

Signaling proteins are tightly regulated spatially and temporally to perform multiple functions. For Cdc42 and other guanosine triphosphatases, the subcellular location of activation is a critical determinant of cell behavior. However, current approaches are limited in their ability to examine the dynamics of Cdc42 activity in living cells. We report the development of a biosensor capable of visualizing the changing activation of endogenous, unlabeled Cdc42 in living cells. With the use of a dye that reports protein interactions, the biosensor revealed localized activation in the trans-Golgi apparatus, microtubule-dependent Cdc42 activation at the cell periphery, and activation kinetics precisely coordinated with cell extension and retraction.

Actins↗

PubMed2003

Melanoma cell migration to type IV collagen requires activation of NF-kappaB.

Chemotaxis is the consequence of environmental factors engaging their receptors to initiate signaling cascades. However, the biochemical mechanisms controlling this phenomenon are not clear. We employed an in vitro modified Boyden 48-well chemotaxis migration system to characterize the role of signal transducers in type IV collagen (CIV) induced A2058 human melanoma cell migration. Using specific pharmacological inhibitors and a series of dominant-negative and constitutively active signaling proteins, we show that Ras and Rac GTPases, PI-3K, and PKC participate in cell migration mediated by beta1 integrins. Collagen also induces a time- dependent degradation of IkappaB-alpha and an increase in nuclear translocation of NF-kappaB which is dependent on PKC pathway. More importantly, collagen-stimulated melanoma cell migration directly correlated with an increase in NF-kappaB transactivation. Furthermore, CIV induced an increase in beta1 integrin mRNA levels. Specific NF-kappaB inhibitors Helenalin and SN-50 inhibited melanoma cell migration to collagen, indicating a novel requirement for NF-kappaB transactivation in cell chemotaxis mediated by beta1 integrin signals. These results describe signal transduction events that are initiated by type IV collagen through beta1 integrins and demonstrate an important role for NF-kappaB in regulating melanoma chemotaxis.

Base Sequence↗

PubMed2002

Advances in molecular labeling, high throughput imaging and machine intelligence portend powerful functional cellular biochemistry tools.

Cellular behavior is complex. Successfully understanding systems at ever-increasing complexity is fundamental to advances in modern science and unraveling the functional details of cellular behavior is no exception. We present a collection of prospectives to provide a glimpse of the techniques that will aid in collecting, managing and utilizing information on complex cellular processes via molecular imaging tools. These include: 1) visualizing intracellular protein activity with fluorescent markers, 2) high throughput (and automated) imaging of multilabeled cells in statistically significant numbers, and 3) machine intelligence to analyze subcellular image localization and pattern. Although not addressed here, the importance of combining cell-image-based information with detailed molecular structure and ligand-receptor binding models cannot be overlooked. Advanced molecular imaging techniques have the potential to impact cellular diagnostics for cancer screening, clinical correlations of tissue molecular patterns for cancer biology, and cellular molecular interactions for accelerating drug discovery. The goal of finally understanding all cellular components and behaviors will be achieved by advances in both instrumentation engineering (software and hardware) and molecular biochemistry.

Animals↗

PubMed2002

Intracellular calcium changes in rat aortic smooth muscle cells in response to fluid flow.

Vascular smooth muscle cells (VSM) are normally exposed to transmural fluid flow shear stresses, and after vascular injury, blood flow shear stresses are imposed upon them. Since Ca2+ is a ubiquitous intracellular signaling molecule, we examined the effects of fluid flow on intracellular Ca2+ concentration in rat aortic smooth muscle cells to assess VSM responsiveness to shear stress. Cells loaded with fura 2 were exposed to steady flow shear stress levels of 0.5-10.0 dyn/cm2 in a parallel-plate flow chamber. The percentage of cells displaying a rise in cytosolic Ca2+ ion concentration ([Ca2+]i) increased in response to increasing flow, but there was no effect of flow on the ([Ca2+]i) amplitude of responding cells. Addition of Gd3+ (10 microM) or thapsigargin (50 nM) significantly reduced the percentage of cells responding and the response amplitude, suggesting that influx of Ca2+ through ion channels and release from intracellular stores contribute to the rise in ([Ca2+]i) in response to flow. The addition of nifedipine (1 or 10 microM) or ryanodine (10 microM) also significantly reduced the response amplitude, further defining the role of ion channels and intracellular stores in the Ca2+ response.

Animals↗

PubMedDate not supplied

Solute transport to the endothelial intercellular cleft: the effect of wall shear stress.

The interendothelial cleft is the major transport pathway across the endothelium for hydrophilic solutes including albumin and low density lipoprotein. Previous models of arterial wall transport have assumed that the entire endothelial cell surface is available for transport from the fluid (blood) phase. One of the consequences of a cleft-mediated solute uptake mechanism is the limited area available for mass transport. This effect, together with the influence of a predominantly longitudinal cleft orientation in relation to flow, dramatically alters the fluid-phase mass transport characteristics relative to what has been assumed previously in analyzing vascular solute uptake problems. We have used a finite element computational model to simulate fluid phase transport to a longitudinal endothelial cleft under realistic wall shear rate conditions. Our numerical results show reduced dependence of the mass transfer rate on the wall shear rate compared to the classical Leveque solution for mass transport in a cross-flow configuration and confirm the significance of the wall and not the fluid as the limiting resistance to transport of macromolecules.

Animals↗