Gary J. Bassell

Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for β- and γ-actin within cultured cerebrocortical neurons. β-Actin protein was highly enriched within growth cones and filopodia, in contrast to γ-actin protein, which was distributed uniformly throughout the cell. β-Actin protein also was shown to be peripherally localized after transfection of β-actin cDNA bearing an epitope tag. β-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike γ-actin mRNAs, which were restricted to the cell body. The rapid localization of β-actin mRNA, but not γ-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of β-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. β-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of β-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

Neurotrophin-induced transport of a β-actin mRNP complex increases β-actin levels and stimulates growth cone motility

Abstract Neurotrophin regulation of actin-dependent changes in growth cone motility may depend on the signaling of β-actin mRNA transport. Formation of an RNP complex between the β-actin mRNA zipcode sequence and Zipcode Binding Protein 1 (ZBP1) was required for its localization to growth cones. Antisense oligonucleotides to the zipcode inhibited formation of this RNP complex in vitro and the neurotrophin-induced localization of β-actin mRNA and ZBP1 granules. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bidirectional movements of granules in neurites that were inhibited by antisense treatment, as visualized by FRAP analysis. NT-3 stimulation of β-actin protein localization was dependent on the 3′UTR and inhibited by antisense treatment. Growth cones exhibited impaired motility in the presense of antisense. These results suggest a novel mechanism to influence growth cone dynamics involving the regulated transport of mRNA.

Active Transport of the Survival Motor Neuron Protein and the Role of Exon-7 in Cytoplasmic Localization

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deletion and/or mutation of the survival motor neuron protein Gene (SMN1) that results in the expression of a truncated protein lacking the C terminal exon-7. Whereas SMN has been shown to be an important component of diverse ribonucleoprotein (RNP) complexes, its function in neurons is unknown. We hypothesize that the active transport of SMN may be important for neurite outgrowth and that disruption of exon-7 could impair its normal intracellular trafficking. SMN was localized in granules that were associated with cytoskeletal filament systems and distributed throughout neurites and growth cones. Live cell imaging of enhanced green fluorescent protein (EGFP)-SMN granules revealed rapid, bidirectional and cytoskeletal-dependent movements. Exon-7 was necessary for localization of SMN into the cytoplasm but was not sufficient for granule formation and transport. A cytoplasmic targeting signal within exon-7 was identified that could completely redistribute the nuclear protein D-box binding factor 1 into the cytoplasm. Neurons transfected with SMN lacking exon-7 had significantly shorter neurites, a defect that could be rescued by redirecting the exon-7 deletion mutant into neurites by a targeting sequence from growth-associated protein-43. These findings provide the first demonstration of cytoskeletal-based active transport of SMN in neuronal processes and the function of exon-7 in cytoplasmic localization. Such observations provide motivation to investigate possible transport defects or inefficiency of SMN associated RNPs in motor neuron axons in SMA.

mRNA and cytoskeletal filaments.

The localization of some mRNAs to distinct intracellular regions is achieved through interactions of the mRNA with cytoskeletal filaments. RNA-cytoskeletal interactions exist that influence the transport, anchoring and translation of mRNA. Recent analysis of RNA movements in living cells suggests the formation of RNA granules and their active transport along microtubules. The anchoring and translation of mRNA may be mediated by interactions with orthogonal networks of F-actin and elongation factor 1alpha.

Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons

Fragile X syndrome is caused by the absence of the fragile X mental‐retardation protein (FMRP), an mRNA‐binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high‐resolution fluorescence imaging methods to document the presence, motility and activity‐dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F‐actin‐rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental‐retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F‐actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR‐dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.

A predominantly nuclear protein affecting cytoplasmic localization of β-actin mRNA in fibroblasts and neurons

The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47–amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.

Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.

Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.

Association of poly(A) mRNA with microtubules in cultured neurons

The structural basis for the synthesis of specific proteins within distinct intraneuronal compartments is unknown. We studied the distribution of poly(A) mRNA within cultured cerebrocortical neurons using high resolution in situ hybridization to identify cytoskeletal components that may anchor mRNA. After 1 day in culture, poly(A) mRNA was distributed throughout all of the initial neurites, including the axon-like process. At 4 days in culture, poly(A) mRNA was distributed throughout the cell body and dendritic processes, but confined to the proximal segment of the axon. Poly(A) mRNA was bound to the cytoskeleton as demonstrated by resistance to detergent extraction. Perturbation of microtubules with colchicine resulted in a major reduction of dendritic poly(A) mRNA; however, this distribution was unaffected by cytochalasin. Ultrastructural in situ hybridization revealed that poly(A) mRNA and associated ribosomes were excluded from tightly bundled microtubules.

Sunrise at the synapse: the FMRP mRNP shaping the synaptic interface.

Recent studies provide new insight into the mechanistic function of Fragile X Mental Retardation Protein (FMRP), paving the way to understanding the biological basis of Fragile X Syndrome. While it has been known for several years that there are spine defects associated with the absence of the mRNA binding protein FMRP, it has been unclear how its absence may lead to specific synaptic defects that underlie the learning and cognitive impairments in Fragile X. One hypothesis under study is that FMRP may play a key role in the regulation of dendritically localized mRNAs, at subsynaptic sites where regulation of local protein synthesis may influence synaptic structure and plasticity. This review highlights recent progress to identify the specific mRNA targets of FMRP and assess defects in mRNA regulation that occur in cells lacking FMRP. In addition, exciting new studies on Fmr1 knockout mice and mutant flies have begun to elucidate a key role for FMRP in synaptic growth, structure, and long-term plasticity.

Binding proteins for mRNA localization and local translation, and their dysfunction in genetic neurological disease

Neurons utilize mRNA transport and local translation as a means to influence development and plasticity. The molecular mechanisms for this mRNA sorting involve the recognition of cis-acting sequences by distinct mRNA binding proteins that have a dual role, acting in both mRNA transport and translational regulation. Other proteins play a part in the assembly of messenger ribonucleoprotein complexes into transport granules. mRNA binding proteins are crucial targets of phosphorylation signals that regulate local translation. Fragile X syndrome and spinal muscular atrophy have emerged as two genetic neurological diseases that could result, in part, from impaired assembly, localization, and translational regulation of these messenger ribonucleoproteins.