Kimberly E. Forsten

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus–like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region–integral membrane proteins cycle through ER in interphase cells.

Endothelial proteoglycans inhibit bFGF binding and mitogenesis.

Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG‐A and PG‐B. The larger sized fraction (PG‐A) had greater inhibitory activity than did PG‐B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG‐A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG‐B. Both proteoglycan fractions contained perlecan‐like core proteins; however, PG‐A contained an additional core protein (approximately 190 kDa) that was not observed in PG‐B. Both proteoglycan fractions bound bFGF directly, and PG‐A bound a significantly greater relative amount of bFGF than did PG‐B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF‐induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis. J. Cell. Physiol. 172:209–220, 1997.

Insulin-like growth factor (IGF) binding protein-3 regulation of IGF-I is altered in an acidic extracellular environment

While extracellular acidification within solid tumors is well‐documented, how reduced pH impacts regulation of insulin‐like growth factor‐I (IGF‐I) has not been studied extensively. Because IGF‐I receptor binding is affected by IGF binding proteins (IGFBPs), we examined how pH impacted IGFBP‐3 regulation of IGF‐I. IGF‐I binding in the absence of IGFBP‐3 was diminished at reduced pH. Addition of IGFBP‐3 reduced IGF‐I cell binding at pH 7.4 but increased surface association at pH 5.8. This increase in IGF‐I binding at pH 5.8 corresponded with an increase in IGFBP‐3 cell association. This, however, was not due to an increase in affinity of IGFBP‐3 for heparin at reduced pH although both heparinase III treatment and heparin addition reduced IGFBP‐3 enhancement of IGF‐I binding. An increase in IGF‐I binding to IGFBP‐3, though, was seen at reduced pH using a cell‐free assay. We hypothesize that the enhanced binding of IGF‐I at pH 5.8 is facilitated by increased association of IGFBP‐3 at this pH and that the resulting cell associated IGF‐I is IGFBP‐3 and not IGF‐IR bound. Increased internalization and nuclear association of IGF‐I at pH 5.8 in the presence of IGFBP‐3 was evident, yet cell proliferation was reduced by IGFBP‐3 at both pH 5.8 and 7.4 indicating that IGFBP‐3‐cell associated IGF‐I does not signal the cell to proliferate and that the resulting transfer of bound IGF‐I from IGF‐IR to IGFBP‐3 results in diminished proliferation. Solution binding of IGF‐I by IGFBP‐3 is one means by which IGF‐I‐induced proliferation is inhibited. Our work suggests that an alternative pathway exists by which IGF‐I and IGFBP‐3 both associate with the cell surface and that this association inhibits IGF‐I‐induced proliferation.

A Simple Assay for Evaluating Inhibitors of Proteoglycan-Ligand Binding

AbstractProteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions.

Real-time detection of insulin-like growth factor-1 stimulation of the MAC-T bovine mammary epithelial cell line.

Binding of growth factors by cell-surface receptors is an essential means by which cell regulate normaltissue growth and differentiation. Exposure to growth factors is often transient, and our goal was to determine whether short-term exposure to insulin-like growth factor-1 (IGF-1) would lead to activation, assayed as cell proliferation, of mammary epithelial cells. The MAC-T cell line is an immortalized bovine mammary epithelial cell line, chosen as our model mammary cell line because of its known sensitivity to IGF-1. Using the Cytosensor Microphysiometer System, a biosensor capable of measuring extracellular acidification, we were able to measure activation of the cellsowing to IGF-1 addition in real time and found that peak acidification occurred in only 14 min. We show that this rapid response to IGF-1 is dose dependent and specific for IGF-1. A significant increase in [3H]thymidine incorporation by cells after a similar short-term exposure to IGF-1 suggests that the measured increase in extracellular acidification following IGF-1 addition is physiologically relevant. This technology offers a new, novel, and rapid means for the study of IGF-1 activity, as well as the screening of IGF-1 inhibitors, in mammary epithelial cells.

Influence of surface chemistry on particle-particle aggregation as measured by a Coulter Counter in the low concentration regime

The link between particle concentration, surface properties, and particle aggregation has been investigated for polystyrene latex in the low particle concentration regime, volume fractions less than 1 × 10-6. The particles used in this model study were 9.14-μm diameter polystyrene beads, approximately the same size as red blood cells, using a Coulter Counter with a 100-μm aperture as a gauge of clustering. The lower concentrations of beads displayed the same trend as reported in other publications. Albumin and fibrinogen were then attached to the beads and the effect of non-specific binding by the proteins on particle agglomeration was studied. Albumin showed little effect on the number of aggregates encountered whereas fibrinogen increased the number of aggregates, indicating that stronger particle interactions occurred between the fibrinogen coated particles. The addition of stearic acid had no marked effect on the aggregation properties of the beads, coated or uncoated. The addition of sodium stearate, however, increased the number of doublets, triplets, and multiplets formed. These results may be relevant to adhesion of cells. Our approach is a plausible way to consider the roles that proteins and fatty acid molecules have on the relative binding potential without the constraints of using live cells.

IGF-I stimulation of extracellular acidification is not linked to cell proliferation for autocrine cells.

Insulin-like growth factor-I (IGF-I) increases extracellular acidification rate (ECAR), a measure correlated with proliferation for nonautocrine cells. To evaluate the effect of autocrine IGF-I secretion on cell responsiveness, a cell line that secretes IGF-I was tested. SV40-IGF=I cells also registered concentration-dependent increases in ECAR; however, unlike the parental cell line, signal attenuation upon repeat challenges was not evident. Furthermore, SV40-IGF-I cells did not proliferate in response to IGF-I. We investigated if lack of proliferation was due to differences in the protocols, of the assays ([3H]thymidine incorporation and microphysiometry). We identified three key differences in the protocols: surface substrate, cell density, and fluid residence time. We found no increase in [3H]thymidine incorporation for cells on either tissueculture plastic or polycarbonate transwells. Control levels of [3H]thymidine incorporation were cell-density-dependent, but IGF-I did not increase proliferation at any density studied. Finally, we investigated IGF-I stimulation for cells under microphysiometer flow conditions and found no proliferative response to IGF-I. We found that the cells do respond to IGF-I with increased amino acid uptake. These data suggest that IGF-I signaling is operational in the SV40-IGF-I cells, but the transduction pathway for IGF-I-induced proliferation is compromised, despite the fact that these cells respond to fetal bovine serum with increased growth. Ongoing studies are focused on identifying which elements in the signaling cascade are altered by autocrine scretion of IGF-I.

A particle adhesion perspective on metastasis

Abstract One area of interest in bioadhesion that has not been emphasized within the adhesion science community relates to the disaggregation of cells which occurs when cancer metastases arise. Metastasis involves the distribution of cancerous tumor cells from a large localized tumor. The resulting spatial separation of the cancerous masses makes treatment more difficult. Making use of biochemical and cellular assays, detailed mechanisms for the cell detachment processes involving cadherin cell adhesion molecules have been proposed. This paper reviews proposed mechanisms for metastasis from a cellular adhesion perspective and the testing methodologies that have been utilized. Additional understanding might be gleaned from considering the loss of cell adhesion molecules and the overall disaggregation process from a particle adhesion perspective. Several pertinent theories are presented as well as a brief discussion of areas for future effort.