In vivo measurement of microtubule dynamics using stable isotope labeling with heavy water. Effect of taxanes

Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium ((2)H(2)) from heavy water ((2)H(2)O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of (2)H(2)O labeling in vivo reduced (2)H labeling in polymers while increasing (2)H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.     

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.     

Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCepsilon translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1-induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.     

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.     

Herpes simplex virus virion host shutoff protein is stimulated by translation initiation factors eIF4B and eIF4H

The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species. Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins. However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined. An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae. We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors. This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B. RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern. In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting. These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES.     

A family-based approach to preventing excessive weight gain

Objective: Preventing weight gain in adults and excessive weight gain in children is a high priority. We evaluated the ability of a family‐based program aimed at increasing steps and cereal consumption (for breakfast and snacks) to reduce weight gain in children and adults. Research Methods and Procedures: Families (n = 105) with at least one 8‐ to 12‐year‐old child who was at‐risk‐for‐overweight or overweight (designated as the target child) were recruited for the study. Eighty‐two families were randomly assigned to receive the family‐based intervention and 23 families to the control condition. The 13‐week intervention consisted of specific increases in daily steps (an additional 2000 steps/d) and consumption of 2 servings/d of ready‐to‐eat cereal. Results: The intervention was successful in increasing walking (steps) and cereal consumption. The intervention had positive, significant effects on percentage BMI‐for‐age and percentage body fat for target children and weight, BMI, and percentage body fat for parents. On further analysis, the positive effects of the intervention were seen largely in target girls and moms, rather than in target boys and dads. Discussion: This family‐based weight gain prevention program based on small changes holds promise for reducing excessive weight gain in families and especially in growing overweight children.     

The case of the disappearing bone: Dogs’ understanding of the physical properties of objects

To assess dogs’ memory for an occluded object, a gaze duration procedure was used similar to one often used with nonverbal infants. A bone shaped dog biscuit was placed behind a solid screen that then rotated in the depth plane through an arc front to back. Dogs were shown either of the two test events. In one event (the possible event), the screen rotated until it reached the point at which it would have reached the bone and then stopped (about 120°); in the other event (the impossible event), the screen rotated through a full 180° arc, as though it had passed through the bone. The dogs looked significantly longer at the impossible event. To control for the differential time it took for the screen to move, for a control group, a bone was placed behind the screen and the screen was rotated either 60° or 120° (both possible events). No difference in looking time was found. To control for the movement of the screen through 120° or 180° when both were possible, for a second control group, the bone was placed to the side of the screen rather than behind the screen and the screen was moved 120° or 180°. Again, no significant difference in looking time was found. Results suggest that much like young children, dogs understand the physical properties of an occluded object. That is they appear to understand that an object (such as a screen) should not be able to pass through another object (such as dog bone).     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.