Biomedical subjects

Ken Jacobson

Publications and source records attributed to Ken Jacobson.

7 recordsLinked to original sources

PubMed2003

JNK phosphorylates paxillin and regulates cell migration.

The c-Jun amino-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. Accordingly, its substrates are transcription factors and anti-apoptotic proteins. However, JNK has also been shown to be required for Drosophila dorsal closure, and MAP kinase/ERK kinase kinase 1, an upstream kinase in the JNK pathway, has been shown to be essential for cell migration. Both results imply that JNK is important in cell migration. Here we show that JNK1 is required for the rapid movement of both fish keratocytes and rat bladder tumour epithelial cells (NBT-II). Moreover, JNK1 phosphorylates serine 178 on paxillin, a focal adhesion adaptor, both in vitro and in intact cells. NBT-II cells expressing the Ser 178 --> Ala mutant of paxillin (Pax(S178A)) formed focal adhesions and exhibited the limited movement associated with such contacts in both single-cell-migration and wound-healing assays. In contrast, cells expressing wild-type paxillin moved rapidly and retained close contacts as the predominant adhesion. Expression of Pax(S178A) also inhibited the migration of two other cell lines. Thus, phosphorylation of paxillin by JNK seems essential for maintaining the labile adhesions required for rapid cell migration.

Amino Acid Sequence↗

PubMed2003

Binding of NAP-22, a calmodulin-binding neuronal protein, to raft-like domains in model membranes.

The cholesterol-binding protein NAP-22 is a major component of the detergent-insoluble low-density fraction of rat brain. In this study, we found, using fluorescence microscopy, that native NAP-22, but not a demyristoylated form, binds to cholesterol-rich raft-like domains in planar-supported monolayers and remains bound after nonionic detergent extraction. NAP-22 also protects the cholesterol-rich domains during extraction by methyl-beta-cyclodextrin. The lateral mobility of this protein is much lower than that of other raft components in model membranes, suggesting that both cholesterol binding and inter-NAP-22 interactions markedly reduce its lateral diffusion. This study suggests that NAP-22 binding may be employed to image cholesterol-rich regions, such as caveolae/rafts, on the plasma membrane of cells, and preliminary efforts in that direction are presented.

3T3 Cells↗

PubMed2002

Phospholipids undergo hop diffusion in compartmentalized cell membrane.

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

Actins↗

PubMed2002

A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains.

The surface membrane of cells is studded with morphologically distinct regions, or domains, like microvilli, cell-cell junctions, and coated pits. Each of these domains is specialized for a particular function, such as nutrient absorption, cell-cell communication, and endocytosis. Lipid domains, which include caveolae and rafts, are one of the least understood membrane domains. These domains are high in cholesterol and sphingolipids, have a light buoyant density, and function in both endocytosis and cell signaling. A major mystery, however, is how resident molecules are targeted to lipid domains. Here, we propose that the molecular address for proteins targeted to lipid domains is a lipid shell.

Animals↗

PubMed2002

Relationship of lipid rafts to transient confinement zones detected by single particle tracking.

We examined the physical and chemical characteristics of transient confinement zones (TCZs) that are detected in single particle trajectories of molecules moving within the membrane of C3H 10T1/2 murine fibroblasts and their relationship to "rafts." We studied the lateral movement of different membrane molecules thought to partition to varying degrees into or out of the putative lipid domains known as rafts. We found that lipid analogs spend significantly less time in TCZs compared with Thy-1, a glycosylphosphatidylinositol-anchored protein, and GM1, a glycosphingolipid. For Thy-1, we found that zone abundance was markedly reduced by cholesterol extraction, suggesting that a major source of the observed temporary confinement is related to the presence of raft domains. More detailed analysis of particle trajectories reveals that zones can be revisited even tens of seconds after the original escape and that diffusion within the zones is reduced by a factor of approximately 2, consistent with the zone being a cholesterol-rich liquid-ordered phase. Surprisingly, transient confinement was not strongly temperature dependent. Overall, our data demonstrate that there are raft-related domains present in certain regions of the plasma membrane of C3H cells, which can persist for tens of seconds.

Animals↗

PubMed2002

Microscope-based techniques to study cell adhesion and migration.

Modern light microscopy has evolved to provide a variety of quantitative imaging techniques and also the capability to perturb structure-function relationships in living cells. The advances have been especially useful in the study of cell adhesion and migration. This review will focus on how such microscopy-based techniques can be useful in situ to study the molecular interactions and dynamics, to locally perturb actin-based structures and to measure the traction forces exerted by motile cells.

Actins↗

PubMed2002

Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins.

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.

3T3 Cells↗