Heterotrimeric G-proteins mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review we briefly summarize what is known about the regulation, signaling and physiological functions of G-proteins. We then focus on a few less explored areas such as regulation of G-proteins by non-GPCRs, and the physiological functions of G-proteins that can not be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated, and the specificity of G-protein interactions with their regulators.
Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors.
Aneuploidy – chromosome instability leading to incorrect chromosome number in dividing cells – can arise from defects in centrosome duplication, bipolar spindle formation, kinetochore-microtubule attachment, chromatid cohesion, mitotic checkpoint monitoring, or cytokinesis. As most tumors show some degree of aneuploidy, mechanistic understanding of these pathways has been an intense area of research to provide potential therapeutics. Here, we present a mechanism for aneuploidy in fission yeast based on spindle pole microtubule defocusing by loss of kinesin-14 Pkl1, leading to kinesin-5 Cut7-dependent aberrant long spindle microtubule minus end protrusions that push the properly segregated chromosomes to the site of cell division, resulting in chromosome cut at cytokinesis. Pkl1 localization and function at the spindle pole is mutually dependent on spindle pole-associated protein Msd1. This mechanism of aneuploidy bypasses the known spindle assembly checkpoint that monitors chromosome segregation.
Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at characteristic constant length [1–3]. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules (MTs) and their interactions with motors and MT-associated proteins (MAPs). Spindle length is further proposed to be important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force-balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic force contributors in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and MT bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects.
We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP 3 R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP 3 R. DANGER interacts with IP 3 R in vitro and co-immunoprecipitates with IP 3 R from cellular preparations. DANGER robustly enhances Ca 2؉ -mediated inhibition of IP 3 R Ca 2؉ release without affecting IP 3 binding in microsomal assays and inhibits gating in single-channel recordings of IP 3 R. DANGER appears to allosterically modulate the sensitivity of IP 3 R to Ca 2؉ inhibition, which likely alters IP 3 R-mediated Ca 2؉ dynamics in cells where DANGER and IP 3 R are co-expressed.The inositol 1,4,5-trisphosphate receptor (IP 3 R) 3 is a large, endoplasmic reticulum (ER) resident protein, which is a key regulator of intracellular Ca 2ϩ signaling (1, 2). Inositol 1,4,5-trisphosphate (IP 3 ) is formed in response to the activation of G protein-coupled receptors or receptor tyrosine kinase receptors located in the plasma membrane (1), which elicit IP 3 Rmediated Ca 2ϩ release from ER stores. The IP 3 recognition site of IP 3 R includes amino acids (aa) 225-578 in the N-terminal portion of the protein, while the Ca 2ϩ channel domain comprises ϳ300 aa in the extreme C terminus (2). The IP 3 binding site and the Ca 2ϩ channel are separated by ϳ2,000 aa, providing a large area for interactions with multiple regulatory proteins including calmodulin, chromogranins, glyceraldehyde-3-phosphate dehydrogenase, RACK1, and caldendrin (3). While these proteins regulate IP 3 R function in diverse ways, only two regulators have been shown to influence Ca 2ϩ sensitivity. Cytochrome c, which binds to the extreme C terminus of the IP 3 R, relieves the inhibitory actions of Ca 2ϩ upon the channel (4), and Bcl-XL binding to the C terminus also influences the Ca 2ϩ dependence (5).We have identified a novel vertebrate protein, designated DANGER, which was isolated by yeast two-hybrid analysis with the regulatory region of the IP 3 R as bait. DANGER physiologically binds to IP 3 R and allosterically enhances the potency of Ca 2ϩ -inhibition of IP 3 R-mediated Ca 2ϩ release without affecting ligand binding. EXPERIMENTAL PROCEDURESYeast Two-hybrid Analysis-The Matchmaker3 yeast-2-hybrid system from Clontech (Palo Alto, CA) was employed. AH109 -galactosidase yeast was used. IP 3 R fragments were cloned into pGADT7 (-galactosidase acceptor domain) vector and were screened against a rat brain and human fetal kidney library (Clontech) as per the manufacturer's specifications. Expression of these fragments was determined by Western blotting using antibodies from Clontech, corresponding to the expression vector. Positive clones grew on minimal SD agar (Clontech) lacking adenine, histidine, leucine, and tryptophan and had -galactosidase activity.Calcium Release Measurements and El...
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