Protein Needed for Cell Skeleton Assembly
To propel itself forward, a cell continually assembles actin filaments in the front and disassembles them in the rear. But the precise signals the cell uses to manipulate its actin cytoskeleton are not fully understood.
Adding a major piece to the puzzle, Rong Li, HMS assistant professor of cell biology; Terry Lechler, a graduate student in cell biology; and colleagues have discovered that Type I myosins, a family of proteins, are essential for actin polymerization. Traditionally, these proteins are thought of as molecular motors that transport cargo along actin filaments. Their role in regulating actin dynamics came as a surprise. "These myosins have been known for 30 years, but their function has been a mystery. We are the first in the field to identify this role," says Li.
Li's team has been studying actin assembly in yeast. Daughter yeast cells bud out from mother cells with the help of actin networks. To learn whether Type I myosins have a role in assembling these networks, the researchers first added chemicals to poke holes in the buds. They then added actin and a variety of yeast cell extracts to see if the actin mesh would reassemble. Extracts from yeast lacking two particular myosins, myo3 and myo5, were unable to restore the actin network. Extracts lacking only one of these proteins were as successful as wild type, suggesting a redundant function of the two proteins (see figure).
Delving into the mechanism, the researchers found that these myosins interact with two molecules known to play a role in actin polymerizationbee1p, a protein previously discovered by Li's group, and the Arp2/3 complex.
The study is featured as the cover story of the Jan. 24 Journal of Cell Biology.
+ myosins - myosins
(myo3 or myo5)
(myo3 & myo5)
Yeast cell extracts missing Type I myosins myo3 and myo5 cannot restore actin assembly in budding yeast daughter cells (right). Extracts lacking only one of the proteins can assemble actin as well as extracts from wild type yeast (left), suggesting a redundant function for the two myosins.
Novel Interaction Discovered Between Endocytosis Proteins
For a cell to ingest a receptor-bound hormone or other molecule, adapter proteins in the cell's membrane must bind to the receptor and to clathrin proteins inside the cell. Clathrin assembles along the internal membrane, directing this region to bud off into clathrin-coated vesicles, which transport the molecular cargo to its proper destination.
Ernst ter Haar, HMS research fellow in cell biology; Stephen Harrison, Howard Hughes investigator, HMS professor of biological chemistry and molecular pharmacology, and director of the Armenise Center for Structural Biology; and Tomas Kirchhausen, HMS professor of cell biology at the Center for Blood Research, have been deciphering the details of how clathrin and various adaptor proteins interact. Now they report an interaction that occurs between clathrin and at least two different types of adaptor.
It was already known that adaptors bind to the "foot" of the three-legged clathrin molecule. The research team solved the crystal structure of the foot in 1998, revealing it to be a propeller-like structure with seven blades (see Focus, Nov. 13, 1998). They have now discovered that segments of two adaptor proteins, beta-arrestin 2 and AP-3, bind to the groove between blades 1 and 2.
Since two different adaptor molecules bind to clathrin in the exact same region, the researchers examined the binding sequences more closely. Indeed, beta-arrestin 2
and AP-3 have related clathrin-binding peptides, and the conserved sequences make identical contacts. Suspecting a pattern, the researchers looked for, and found, related highly conserved sequences in eight other adaptor proteins. They say it is likely these "clathrin-box motifs" bind similarly in the blade 1blade 2 groove.
The researchers believe this "peptide-in-groove" interaction may be a mode by which other propeller-containing molecules interact with target proteins. Their study appears in the Feb. 1 Proceedings of the National Academy of Sciences.
New Function Found
For Kidney Disease Gene
Scientists at Massachusetts General Hospital have discovered that the polycystin-1 gene (PKD1), which is mutated in patients with autosomal dominant polycystic kidney disease (ADPKD), is essential for the structural integrity of blood vessels.
ADPKD is the most common single-gene disorder, affecting from 1 in 400 to 1 in 1,000 people. According to the study's leader, M. Amin Arnaout, HMS professor of medicine and chief of nephrology at MGH, "It is the fourth leading cause of chronic renal failure, requiring hemodialysis or transplantation, and costs more than one billion dollars annually."
People with the disease develop renal cysts, which often lead to kidney failure. Vascular abnormalities, such as aneurysms in cerebral vessels, are also common and are a leading cause of sudden death in young patients. Vascular fragility is generally thought to be a secondary effect of hypertension, commonly found in patients with ADPKD. The MGH study, appearing in the Feb. 15 Proceedings of the National Academy of Sciences, is the first to show a direct role for polycystin-1 in blood vessel lesions.
To study polycystin-1's function, Arnaout; Keetae Kim, HMS research fellow in medicine; Iain Drummond, HMS assistant professor of medicine; and colleagues from Genzyme mutated the gene in mice. Approximately halfway through gestation, the mice developed kidney cysts, vascular leaks, edema, and ruptured blood vessels. All mice died before birth, likely because of massive hemorrhages.
Although the precise function of polycystin-1 is not known, it was found to be located between endothelial and epithelial cell junctions. Cellcell junctions play essential roles in cell growth and differentiation, processes that are abnormal in ADPKD.
Other researchers have observed kidney cysts associated with PKD1 mutations in mice but have not found vascular problems. The MGH researchers now believe that variability in the outward manifestation of the disease in humans is determined by the nature of the PKD1 mutation.
Oncogene's Suppression Reverses Leukemia
HMS researchers have discovered that leukemia can be induced and reversed in mice by manipulating the expression of the gene BCR-ABL.The study, appearing in the January Nature Genetics,provides strong evidence that certain types of leukemia can be caused by the expression of a single gene rather than multiple genes, as is thought to be the case in many cancers.
BCR-ABL is a chimeric gene produced by chromosomal translocation. The removal of ABL gene sequences from chromosome 9 and their insertion into the BCR gene on chromosome 22 creates this potent oncogene, which stimulates division of hematopoietic stem cells. The gene is found in the majority of patients with chronic myelogenous leukemia and in some patients with acute lymphoblastic leukemia.
Key to the discovery was the development of a new mouse model. Beth Israel Deaconess Medical Center researchers Claudia Huettner, HMS instructor in medicine; Richard Van Etten, HMS associate professor of genetics (medicine); and Daniel Tenen, HMS professor of medicine, along with Pu Zhang, HMS instructor in medicine at the Center for Blood Research, created the transgenic mice, which only express BCR-ABL if the antibiotic tetracycline is removed from their drinking water.
Tetracycline withdrawal, allowing BCR-ABL expression, resulted in lethal leukemia in all mice within three to six weeks. To test whether the disease could be reversed, mice with advanced leukemia were given tetracycline. Interestingly, white blood cell counts rapidly dropped, reaching normal levels within 48 to 72 hours. Within five days, enlarged lymph nodes completely regressed. Further studies demonstrated that end-stage leukemia could be repeatedly induced and reversed in the same animal by withdrawal and re-administration of tetracycline.
The study's leader, Tenen, is encouraged by his group's findings. He says, "These and other studies suggest that some leukemias may be caused by as little as a single genetic change. Specific therapies directed at these changes, such as the BCR-ABLspecific tyrosine kinase inhibitor STI571, will be very effective in these leukemias."
Briefs by Lorene Leiter