Rules of engagement promote polarity in RNA trafficking

Many cell biological pathways exhibit overall polarity (net movement of molecules in one direction) even though individual molecular interactions in the pathway are freely reversible. The A2 RNA trafficking pathway exhibits polarity in moving specific RNA molecules from the nucleus to localization sites in the myelin compartment of oligodendrocytes or dendritic spines in neurons. The A2 pathway is mediated by a ubiquitously expressed trans-acting trafficking factor (hnRNP A2) that interacts with a specific 11 nucleotide cis-acting trafficking sequence termed the A2 response element (A2RE) found in several localized RNAs. Five different molecular partners for hnRNP A2 have been identified in the A2 pathway: hnRNP A2 itself, transportin, A2RE RNA, TOG (tumor overexpressed gene) and hnRNP E1, each playing a key role in one particular step of the A2 pathway. Sequential interactions of hnRNP A2 with different molecular partners at each step mediate directed movement of trafficking intermediates along the pathway. Specific "rules of engagement" (both and, either or, only if) govern sequential interactions of hnRNP A2 with each of its molecular partners. Rules of engagement are defined experimentally using three component binding assays to measure differential binding of hnRNP A2 to one partner in the presence of each of the other partners in the pathway. Here we describe rules of engagement for hnRNP A2 binding to each of its molecular partners and discuss how these rules of engagement promote polarity in the A2 RNA trafficking pathway. For molecules with multiple binding partners, specific rules of engagement govern different molecular interactions. Rules of engagement are ultimately determined by structural relationships between binding sites on individual molecules. In the A2 RNA trafficking pathway rules of engagement governing interactions of hnRNP A2 with different binding partners provide the basis for polarity of movement of intermediates along the pathway

Kinetochore fiber formation in animal somatic cells : dueling mechanisms come to a draw

Author Posting. © The Author, 2005. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Chromosoma 114 (2005): 310-318, doi:10.1007/s00412-005-0028-2.The attachment to and movement of a chromosome on the mitotic spindle is mediated by the formation of a bundle of microtubules (MTs) that tethers the kinetochore on the chromosome to a spindle pole. The origin of these “kinetochore fibers” (K-fibers) has been investigated for over 125 years. As noted in 1944 by Schrader, there are only three possible ways to form a K-fiber: either it a) grows from the pole until it contacts the kinetochore; b) grows directly from the kinetochore; or c) it forms as a result of an interaction between the pole and the chromosome. Since Schrader’s time it has been firmly established that K-fibers in centrosome-containing animal somatic cells form as kinetochores capture MTs growing from the spindle pole (route a). It is now similarly clear that in cells lacking centrosomes, including plants and many animal oocytes, K-fibers “self-assemble” from MTs generated by the chromosomes (route b). Can animal somatic cells form K-fibers in the absence of centrosomes by the “self-assembly” pathway? In 2000 the answer to this question was shown to be a resounding “yes”. With this result, the next question became whether the presence of a centrosome normally suppresses K-fiber self-assembly, or if this route works concurrently with centrosome-mediated K-fiber formation. This question, too, has recently been answered: observations on untreated live animal cells expressing GFP-tagged tubulin clearly show that kinetochores can nucleate the formation of their associated MTs in the presence of functional centrosomes. The concurrent operation of these two “dueling” routes for forming K-fibers in animals helps explain why the attachment of kinetochores and the maturation of K-fibers occur as quickly as it does on all chromosomes within a cell.The work is sponsored by NIH grant GMS 40198

Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca(2+)- and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways

Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach

International audienc

Stathmin Regulates Centrosomal Nucleation of Microtubules and Tubulin Dimer/Polymer Partitioning

Stathmin is a microtubule-destabilizing protein ubiquitously expressed in vertebrates and highly expressed in many cancers. In several cell types, stathmin regulates the partitioning of tubulin between unassembled and polymer forms, but the mechanism responsible for partitioning has not been determined. We examined stathmin function in two cell systems: mouse embryonic fibroblasts (MEFs) isolated from embryos +/+, +/−, and −/− for the stathmin gene and porcine kidney epithelial (LLCPK) cells expressing stathmin-cyan fluorescent protein (CFP) or injected with stathmin protein. In MEFs, the relative amount of stathmin corresponded to genotype, where cells heterozygous for stathmin expressed half as much stathmin mRNA and protein as wild-type cells. Reduction or loss of stathmin resulted in increased microtubule polymer but little change to microtubule dynamics at the cell periphery. Increased stathmin level in LLCPK cells, sufficient to reduce microtubule density, but allowing microtubules to remain at the cell periphery, also did not have a major impact on microtubule dynamics. In contrast, stathmin level had a significant effect on microtubule nucleation rate from centrosomes, where lower stathmin levels increased nucleation and higher stathmin levels reduced nucleation. The stathmin-dependent regulation of nucleation is only active in interphase; overexpression of stathmin-CFP did not impact metaphase microtubule nucleation rate in LLCPK cells and the number of astral microtubules was similar in stathmin +/+ and −/− MEFs. These data support a model in which stathmin functions in interphase to control the partitioning of tubulins between dimer and polymer pools by setting the number of microtubules per cell

Global Regulation of the Interphase Microtubule System by Abundantly Expressed Op18/Stathmin

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer–polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer–polymer partitioning, enables Op18 to exert global regulation of the microtubule system

Aneugenic Activity of Op18/Stathmin Is Potentiated by the Somatic Q18→E Mutation in Leukemic Cells

Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18→E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18→E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18→E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors