scholarly journals Doublecortin facilitates the elongation of the somatic Golgi apparatus into proximal dendrites

2021 ◽  
pp. mbc.E19-09-0530
Author(s):  
Peijun Li ◽  
Luyao Li ◽  
Binyuan Yu ◽  
Xinye Wang ◽  
Qi Wang ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Underlying Mechanism ◽  
Neurite Length ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Cortical Malformations ◽  
Subcortical Band Heterotopia ◽  
Associated Proteins ◽  
Band Heterotopia

Mutations in the doublecortin ( DCX) gene, which encodes a microtubule-binding protein, cause human cortical malformations, including lissencephaly and subcortical band heterotopia. A deficiency in DCX and doublecortin-like kinase 1 (DCLK1), a functionally redundant and structurally similar cognate of DCX, decreases neurite length and increases the number of primary neurites directly arising from the soma. The underlying mechanism is not completely understood. In this study, the elongation of the somatic Golgi apparatus into proximal dendrites, which have been implicated in dendrite patterning, was significantly decreased in the absence of DCX/DCLK1. Phosphorylation of DCX at S47 or S327 was involved in this process. DCX deficiency shifted the distribution of CLASP2 proteins to the soma from the dendrites. In addition to CLASP2, dynein and its co-factor JIP3 were abnormally distributed in DCX-deficient neurons. The association between JIP3 and dynein was significantly increased in the absence of DCX. Downregulation of CLASP2 or JIP3 expression with specific shRNAs rescued the Golgi phenotype observed in DCX-deficient neurons. We conclude that DCX regulates the elongation of the Golgi apparatus into proximal dendrites through microtubule-associated proteins and motors.

scholarly journals Prodigiosin-Emerged PI3K/Beclin-1-Independent Pathway Elicits Autophagic Cell Death in Doxorubicin-Sensitive and -Resistant Lung Cancer

2018 ◽  
Vol 7 (10) ◽  
pp. 321 ◽  
Author(s):  
Wei-Jun Chiu ◽  
Shian-Ren Lin ◽  
Yu-Hsin Chen ◽  
May-Jwan Tsai ◽  
Max Leong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Mammalian Target Of Rapamycin ◽  
Underlying Mechanism ◽  
Beclin 1 ◽  
Class Iii ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Anti Tumor Activity

Prodigiosin (PG) belongs to a family of prodiginines isolated from gram-negative bacteria. It is a water insoluble red pigment and a potent proapoptotic compound. This study elucidates the anti-tumor activity and underlying mechanism of PG in doxorubicin-sensitive (Dox-S) and doxorubicin-resistant (Dox-R) lung cancer cells. The cytotoxicity and cell death characteristics of PG in two cells were measured by MTT assay, cell cycle analysis, and apoptosis/autophagic marker analysis. Then, the potential mechanism of PG-induced cell death was evaluated through the phosphatidylinositol-4,5-bisphosphate 3-kinase-p85/Protein kinase B /mammalian target of rapamycin (PI3K-p85/Akt/mTOR) and Beclin-1/phosphatidylinositol-4,5-bisphosphate 3-kinase-Class III (Beclin-1/PI3K-Class III) signaling. Finally, in vivo efficacy was examined by intratracheal inoculation and treatment. There was similar cytotoxicity with PG in both Dox-S and Dox-R cells, where the half maximal inhibitory concentrations (IC50) were all in 10 μM. Based on a non-significant increase in the sub-G1 phase with an increase of microtubule-associated proteins 1A/1B light chain 3B-phosphatidylethanolamine conjugate (LC3-II), the cell death of both cells was categorized to achieve autophagy. Interestingly, an increase in cleaved-poly ADP ribose polymerase (cleaved-PARP) also showed the existence of an apoptosis-sensitive subpopulation. In both Dox-S and Dox-R cells, PI3K-p85/Akt/mTOR signaling pathways were reduced, which inhibited autophagy initiation. However, Beclin-1/PI3K-Class III downregulation implicated non-canonical autophagy pathways were involved in PG-induced autophagy. At completion of the PG regimen, tumors accumulated in the mice trachea and were attenuated by PG treatment, which indicated the efficacy of PG for both Dox-S and Dox-R lung cancer. All the above results concluded that PG is a potential chemotherapeutic agent for lung cancer regimens regardless of doxorubicin resistance.

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 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 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 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.

scholarly journals Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

2004 ◽  
Vol 384 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Josefa ANDRADE ◽  
Sandy Timm PEARCE ◽  
Hu ZHAO ◽  
Margarida BARROSO
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Independent Manner ◽  
Microtubule Associated Proteins ◽  
Binding Partners ◽  
Ef Hand ◽  
Associated Proteins ◽  
Microsomal Membranes ◽  
Terminal Amino

Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30–100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35–40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a KD of ∼0.5 μM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule–membrane interactions by affecting the p22–microtubule, but not the p22–membrane, association.

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