The retinotectal map is the best characterized model system to study how axons respond to guidance cues during the formation of the nervous system. Recent studies have shown that the critical event in forming this map is topographic-specific axon branching. To elucidate the in vivo role of the repulsive cue ephrin-A5 in this event, we used chromophore-assisted laser inactivation (CALI) to generate acute loss of ephrin-A5 function in localized areas of the posterior tectum of chick embryos in ovo and analyzed the resulting changes of retinal projections during initial outgrowth (E11) and when retinal axons arborize in the deep layers in the tectum (E12). We confirmed that ephrin-A5 functions to restrict initial axon outgrowth at E11. At E12, CALI of ephrin-A5 did not affect the extent of axon outgrowth on the tectal surface but instead caused ectopic arborization posterior to the topographically correct site in deeper layers of the tectum. This shows that ephrin-A5 restricts arborization during this critical process for developing the retinotopic map. CALI provides an approach to inactivate in vivo function in higher vertebrates with high temporal and spatial specificity that may have wide application.
Heat shock proteins (hsps) are versatile molecular chaperones that are responsible for many cellular functions including proper folding, oligomeric assembly, activation, and transport of proteins. Most of the known roles for hsps involve intracellular proteins and processes. Mounting evidence suggests that hsps are present and function in the extracellular space. Hsp90alpha was recently found on the surface and in conditioned media of HT-1080 fibrosarcoma cells. Here it acts as a molecular chaperone that assists in the activation of matrix metalloproteinase-2 (MMP2), leading to increased tumor invasiveness. Few other extracellular substrates of hsp90 have been identified, but several independent observations of extracellular hsp90 suggest that this protein may be important for both normal physiology and disease states. Hsp90 typically works in a complex of associated proteins, and some of these proteins have also been observed extracellularly. Here we show that some of these components, including hsp90 organizing protein (hop) and p23, are also found in HT-1080 conditioned media supporting the notion that hsp90 complexes function in invasiveness. These findings suggest a wide-ranging phenomenon of extracellular molecular chaperoning that could have implications for biological processes and disease.
Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC‐12 cell model to explore the biological role of LN–PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN‐coated culture plaques. Furthermore, chromophore‐assisted laser inactivation of cell surface PrPc perturbs LN‐induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.
“Extracellular” Heat Shock Protein-90 (Hsp90) was initially reported in the 1970s but was not formally recognized until 2008 at the 4th International Conference on The Hsp90 Chaperone Machine (Monastery Seeon, Germany). Studies presented under the topic of “extracellular Hsp90 (eHsp90)” at the conference provided direct evidence for eHsp90’s involvement in cancer invasion and skin wound healing. Over the past 15 years, studies have focused on the secretion, action, biological function, therapeutic targeting, preclinical evaluations, and clinical utility of eHsp90 using wound healing, tissue fibrosis, and tumour models both in vitro and in vivo. eHsp90 has emerged as a critical stress-responding molecule targeting each of the pathophysiological conditions. Despite the studies, our current understanding of several fundamental questions remains little beyond speculation. Does eHsp90 indeed originate from purposeful live cell secretion or rather from accidental dead cell leakage? Why did evolution create an intracellular chaperone that also functions as a secreted factor with reported extracellular duties that might be (easily) fulfilled by conventional secreted molecules? Is eHsp90 a safer and more optimal drug target than intracellular Hsp90 chaperone? In this review, we summarize how much we have learned about eHsp90, provide our conceptual views of the findings, and make recommendations on the future studies of eHsp90 for clinical relevance.
It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in approximately 18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.
Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.
