During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% ؎ 3.5% to 80.7% ؎ 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.A key requirement for viral infection or gene transfer by viral or nonviral vectors is the efficient transport of genetic material from the cell surface to the nucleus. The capsid of the adenovirus (Ad) is programmed to achieve vectorial transport of the Ad genome to the nucleus of a target cell by facilitating a sequential progression of protein-protein interactions between capsid proteins and cellular proteins. The initial interaction is the binding step involving the high-affinity interaction of the Ad fiber with the coxsackie-Ad receptor and the interaction of the Ad penton base with integrins (3,6,12,24,65,77,78). The interaction of Ad with its receptors enables Ad to undergo receptor-mediated endocytosis (74). After entering the cell, Ad lyses the endosomal membrane and escapes to the cytosol where it interacts with cytoplasmic dynein and microtubules, resulting in movement of Ad toward the nucleus (10,31,32,36,41,60,61). Finally, studies have indicated that there is a direct interaction between the Ad capsid and proteins in the nuclear envelope (9, 10, 66, 79).The focus of the present study is the interaction of Ad with cytoplasmic dynein and microtubules. Electron microscopy studies showed Ad in close association with microtubules during infection (10, 41). Microt...
The precise orchestration of synaptic differentiation is critical for efficient information exchange in the nervous system. The nerve-muscle synapse forms in response to agrin, which is secreted from the motor nerve terminal and induces the clustering of acetylcholine receptors (AChRs) and other elements of the postsynaptic apparatus on the subjacent muscle cell surface. In view of the highly restricted spatial localization and the plasticity of neuromuscular junctions, it seems likely that synapse formation and maintenance are regulated by additional, as-yet-unidentified factors. Here, we tested whether neurotrophins modulate the agrin-induced differentiation of postsynaptic specializations. We show that both brain-derived neurotrophic factor ( The formation, maintenance, and plasticity of synaptic connections is essential for the proper functioning of the nervous system. A hallmark of fast synapses is the precise spatial registration of the nerve terminal and postsynaptic apparatus. This alignment has been long appreciated in nerve-muscle synapses (1) and has also been demonstrated in a wide range of neuronal synapses (2). Synaptic structure also is tightly regulated: a large number of the synaptic connections initially formed in both the central nervous system (CNS) and the periphery are pruned by the process of synapse elimination.
During infection, adenovirus-associated virus (AAV) undergoes microtubule-dependent retrograde transport as part of a program of vectorial transport of viral genome to the nucleus. A microtubule binding assay was used to evaluate the hypothesis that cytoplasmic dynein mediates AAV interaction with microtubules. Binding of AAV serotype 2 (AAV2) was enhanced in a nucleotide-dependent manner by the presence of total cellular microtubule-associated proteins (MAPs) but not cytoplasmic dynein-depleted MAPs. Excess AAV2 capsid protein prevented microtubule binding by AAV serotypes 2, 5, and rh.10, as well as adenovirus serotype 5, indicating that similar binding sites are used by these viruses.
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