scholarly journals Cytoplasmic Dynein Mediates Adenovirus Binding to Microtubules

2004 ◽  
Vol 78 (18) ◽  
pp. 10122-10132 ◽  
Author(s):  
Samir A. Kelkar ◽  
K. Kevin Pfister ◽  
Ronald G. Crystal ◽  
Philip L. Leopold
Keyword(s):  
Molecular Motor ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Bovine Brain ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Primary Mode ◽  
Cell Lysate ◽  
Brain Maps ◽  
Associated Proteins

ABSTRACT 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.

scholarly journals A Common Mechanism for Cytoplasmic Dynein-Dependent Microtubule Binding Shared among Adeno-Associated Virus and Adenovirus Serotypes

2006 ◽  
Vol 80 (15) ◽  
pp. 7781-7785 ◽  
Author(s):  
Samir Kelkar ◽  
Bishnu P. De ◽  
Guangping Gao ◽  
James M. Wilson ◽  
Ronald G. Crystal ◽  
...  
Keyword(s):  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Common Mechanism ◽  
Dependent Manner ◽  
Adeno Associated Virus ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Serotype 5 ◽  
Vectorial Transport

ABSTRACT 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.

scholarly journals Functionally distinct isoforms of dynactin are expressed in human neurons.

1996 ◽  
Vol 7 (8) ◽  
pp. 1167-1180 ◽  
Author(s):  
M K Tokito ◽  
D S Howland ◽  
V M Lee ◽  
E L Holzbaur
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Motif ◽  
Binding Assays ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Human Neurons ◽  
Alternative Mrna Splicing ◽  
Associated Proteins ◽  
Dynactin Complex

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

scholarly journals Reconstitution of dynein transport to the microtubule plus end by kinesin

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Anthony J Roberts ◽  
Brian S Goodman ◽  
Samara L Reck-Peterson
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Directed Motion ◽  
Microtubule Associated Proteins ◽  
Spatial Regulation ◽  
Intracellular Movement ◽  
Associated Proteins ◽  
Directed Motility ◽  
Dynamic Microtubule

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.

scholarly journals Competition between microtubule-associated proteins directs motor transport

2017 ◽  
Author(s):  
Brigette Y. Monroy ◽  
Danielle L. Sawyer ◽  
Bryce E. Ackermann ◽  
Melissa M. Borden ◽  
Tracy C. Tan ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Inhibitory Effect ◽  
Microtubule Associated Proteins ◽  
Motor Transport ◽  
Branch Formation ◽  
Microtubule Motor ◽  
Associated Proteins

Within cells, numerous motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule lattice. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of microtubule motor transport is unknown. Here, we examine the relationship between MAP7 and tau due to their antagonistic effects on neuronal branch formation and kinesin motility in vivo1–8. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. In striking contrast to the inhibitory effect of tau, MAP7 promotes kinesin-based transport in vivo and strongly enhances kinesin-1 binding to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. In contrast, both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs exhibit differential control over distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of molecular motor activity.

Microtubule motors and other maps

1990 ◽  
Vol 48 (3) ◽  
pp. 1-1
Author(s):  
Richard B. Vallee
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Structural Components ◽  
Microtubule Associated Proteins ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
The Subject ◽  
Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

scholarly journals Domains of Neuronal Microtubule-associated Proteins and Flexural Rigidity of Microtubules

1997 ◽  
Vol 138 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
Harald Felgner ◽  
Rainer Frank ◽  
Jacek Biernat ◽  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Flexural Rigidity ◽  
Microtubule Binding ◽  
Fourfold Increase ◽  
Microtubule Associated Proteins ◽  
Relative Contribution ◽  
Low Concentrations ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
And Function

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at ∼20% saturation with full-length tau, a microtubule exhibits &gt;80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

scholarly journals Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

2014 ◽  
Vol 89 (5) ◽  
pp. 2777-2791 ◽  
Author(s):  
Jingjing Cao ◽  
Cui Lin ◽  
Huijuan Wang ◽  
Lun Wang ◽  
Niu Zhou ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Molecular Mechanisms ◽  
Molecular Motor ◽  
Direct Interaction ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Virus Transport ◽  
Antiviral Therapies ◽  
Early Endosomes ◽  
Replication Sites

ABSTRACTMicrotubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo.IMPORTANCEIncoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings highlight a mechanism whereby circoviruses recruit dynein for transport to the nucleus via the dynein/microtubule machinery.

scholarly journals A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei

1992 ◽  
Vol 117 (1) ◽  
pp. 95-103 ◽  
Author(s):  
A Hemphill ◽  
M Affolter ◽  
T Seebeck
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Binding Motif ◽  
Amino Acid Repeat ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Repeat Units ◽  
Associated Proteins

The major component of the cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei is a membrane skeleton which consists of a single layer of tightly spaced microtubules. This array encloses the entire cell body, and it is apposed to, and connected with, the overlying cell membrane. The microtubules of this array contain numerous microtubule-associated proteins. Prominent among those is a family of high molecular weight, repetitive proteins which consist to a large extent of tandemly arranged 38-amino acid repeat units. The binding of one of these proteins, MARP-1, to microtubules has now been characterized in vitro and in vivo. MARP-1 binds to microtubules via tubulin domains other than the COOH-termini used by microtubule-associated proteins from mammalian brain, e.g., MAP2 or Tau. In vitro binding assays using recombinant protein, as well as transfection of mammalian cell lines, have established that the repetitive 38-amino acid repeat units represent a novel microtubule-binding motif. This motif is very similar in length to those of the mammalian microtubule-associated proteins Tau, MAP2, and MAP-U, but both its sequence and charge are different. The observation that the microtubule-binding motifs both of the neural and the trypanosomal proteins are of similar length may reflect the fact that both mediate binding to the same repetitive surface, the microtubule, while their sequence and charge differences are in agreement with the observation that they interact with different domains of the tubulins.

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