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3. Characterization of a β-Actin mRNA Zipcode-Binding Protein

Author

Ross, Anthony F., Oleynikov, Yuri, Kislauskis, Edward H., Taneja, Krishan L., and Singer, Robert H.

Abstract

Localization of β-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3′ untranslated region of the mRNA, including a 54-nucleotide element which has been termed the “zipcode” (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441–451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize β-actin mRNA.

Published
1997
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4. Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons.

Author

Tiruchinapalli DM, Oleynikov Y, Kelic S, Shenoy SM, Hartley A, Stanton PK, Singer RH, and Bassell GJ

Subjects
Actins biosynthesis, Actins genetics, Animals, Astrocytes cytology, Cells, Cultured, Coculture Techniques, Cytoplasmic Granules metabolism, Cytoplasmic Granules ultrastructure, Dendrites metabolism, Excitatory Amino Acid Antagonists pharmacology, Green Fluorescent Proteins, Hippocampus cytology, Hippocampus embryology, Internet, Luminescent Proteins genetics, Microscopy, Fluorescence methods, Neurons cytology, Potassium Chloride pharmacology, Protein Transport physiology, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Rats, Receptors, N-Methyl-D-Aspartate antagonists & inhibitors, Receptors, N-Methyl-D-Aspartate metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Video Recording, Actins metabolism, Cell Surface Extensions metabolism, Hippocampus metabolism, Neurons metabolism, RNA-Binding Proteins metabolism
Abstract

RNA binding proteins may be important mediators of the activity-dependent transport of mRNAs to dendritic spines of activated synapses. We used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its dynamic movements in response to KCl-induced depolarization at high spatial and temporal resolution. With the use of immunofluorescence, image deconvolution, and three-dimensional reconstruction, ZBP1 was localized in the form of granules that were distributed in dendrites, spines, and subsynaptic sites. KCl depolarization increased the dendritic localization of ZBP1 that was not attributed to an increase in ZBP1 expression. Live cell imaging of single cells before and after perfusion of KCl revealed the rapid and directed efflux of ZBP1 granules from the cell body into dendrites in a proximo-distal gradient. High-speed imaging of enhanced green fluorescence protein-ZBP1 granules revealed rapid anterograde and retrograde movements in dendrites as well as dynamic movements in dendritic spines. A population of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association in dendrites was increased by KCl depolarization. The NMDA receptor antagonist AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and inhibited the KCl-induced transport of ZBP1. The activity-dependent trafficking of ZBP1 and its dynamic movements within dendritic spines provide new evidence to implicate RNA binding proteins as regulators of mRNA transport to activated synapses in response to synaptic activity.

Published
2003

5. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.

Author

Wang W, Wyckoff JB, Frohlich VC, Oleynikov Y, Hüttelmaier S, Zavadil J, Cermak L, Bottinger EP, Singer RH, White JG, Segall JE, and Condeelis JS

Subjects
Adenocarcinoma metabolism, Animals, Cell Adhesion Molecules biosynthesis, Cell Adhesion Molecules genetics, Cell Movement genetics, Cell Movement physiology, Cell Survival genetics, Cell Survival physiology, Chemotaxis physiology, Collagen metabolism, Cytoskeleton metabolism, Cytoskeleton physiology, Disease Models, Animal, Extracellular Matrix metabolism, Extracellular Matrix physiology, Gene Expression, Gene Expression Profiling, Mammary Neoplasms, Experimental metabolism, Microscopy, Confocal, Neoplasm Invasiveness, Neoplasm Metastasis, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Neoplastic Stem Cells physiology, Rats, Rats, Inbred F344, Reverse Transcriptase Polymerase Chain Reaction, Tumor Cells, Cultured, Adenocarcinoma genetics, Adenocarcinoma pathology, Mammary Neoplasms, Experimental genetics, Mammary Neoplasms, Experimental pathology
Abstract

We have developed animal models of breast cancer that allow the direct examination of the behavior of individual green fluorescent protein-expressing carcinoma cells in live nonmetastatic and metastatic primary tumors in situ. We have combined this model with multiphoton microscopy to image differences in cell behavior within the primary tumor. Differences in cell behavior between nonmetastatic and metastatic cells in culture and within live primary tumors were correlated with results from cDNA microarray analyses to identify potentially important genetic determinants for breast cancer invasion and metastasis. Using multiphoton microscopy, we found five major differences in carcinoma cell behavior between the nonmetastatic and metastatic primary breast tumors involving extracellular matrix, cell motility, and chemotaxis. Behavioral differences were correlated with seven categories of molecules that were differentially expressed and related to these behaviors. We have found that extracellular matrix composition, actin nucleation factors, molecules involved in mechanical stability and survival, and cell polarity and chemotaxis showed large and consistent differences in gene expression. We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior.

Published
2002

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