scholarly journals Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein

2019 ◽  
Author(s):  
Qingzhou Feng ◽  
Allison M. Gicking ◽  
William O. Hancock
Keyword(s):  
Adaptor Protein ◽  
Cytoplasmic Dynein ◽  
Quantitative Agreement ◽  
Activation Model ◽  
Bidirectional Transport ◽  
Processive Stepping ◽  
And Diffusion ◽  
Remaining Time ◽  
Allosteric Activator

AbstractCytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used Interferometric Scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB - kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity, in quantitative agreement with the increase in diffusivity seen in isolated DDB. The data suggest a DDB activation model in which engagement of dynactin p150 with the microtubule promotes dynein processivity, serves as an allosteric activator of dynein, and enhances processive minus-end motility during intracellular bidirectional transport.TOC HighlightDynein-dynactin-BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1 – DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.

scholarly journals A new calcium-activated dynein adaptor protein, CRACR2a, regulates clathrin-independent endocytic traffic in T cells

2018 ◽  
Author(s):  
Yuxiao Wang ◽  
Walter Huynh ◽  
Taylor D. Skokan ◽  
Ronald D. Vale
Keyword(s):  
T Cells ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cytoplasmic Dynein ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Organizing Center ◽  
Activated T Cells

AbstractCytoplasmic dynein is a microtubule minus-end-directed motor that transports numerous intracellular cargoes. Mammalian dynein transport is initiated by coiled-coil adaptor proteins that 1) join dynein and its co-factor dynactin into a complex capable of processive motility, and 2) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, which have a coiled-coil adaptor domain, a pair of EF hands, and a Rab GTPase domain fused into a single polypeptide. We find that CRACR2a-mediated dynein-dynactin motility is activated by calcium in vitro and in cells. In activated T cells, CRACR2a localizes to clathrin-independent endosomes that require microtubule-based transport to detach from the actin cortex and travel towards the microtubule organizing center. Together these results represent the first known examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in regulation of endocytic trafficking in T cells.

Analyzing kinesin and cytoplasmic-dynein driven movement with nanometer precision

1989 ◽  
Vol 47 ◽  
pp. 833-833
Author(s):  
B.J. Schnapp ◽  
J. Gelles ◽  
E. Steuer ◽  
M. P. Sheetz
Keyword(s):  
Correlation Function ◽  
Standard Deviation ◽  
Relative Position ◽  
Glass Surface ◽  
Cross Correlation ◽  
Video Sequence ◽  
Cytoplasmic Dynein ◽  
Bidirectional Transport ◽  
Dic Microscopy

Kinesin and cytoplasmic dynein are microtubule-based, force-generating ATPases thought to be involved in the bidirectional transport of vesicular organelles along microtubules. In vitro, kinesin and dynein promote movement of 0.1 - 0.2μm plastic beads toward the plus and minus ends, respectively, of microtubules. In these systems, the proteins are thought to remain fixed, by nonspecific absorption, to the bead surface. The motion of the bead on the microtubule is detected by video enhanced, DIC microscopy, and reflects the movement of the motor protein along the microtubule.To quantitate bead motion, a method was devised for tracking the position of kinesin or dynein coated beads moving along microtubules. The method involves 1) cross-correlation of a template bead image (the kernal) with each of the frames in a video sequence, followed by 2) calculation of the centroid of the cross-correlation function for each of the frames in the sequence. The centroid provides a measure of bead position. Application of this method to the measurement of the relative position of two stationary beads adsorbed nonspecifically to the glass surface indicates that the standard deviation of the measured horizontal and vertical coordinates can be less than 0.5 nm. More recently, we have determined that centroid calculation of the unprocessed bead image (i.e. without cross-correlation) can provide a measure of bead position with standard deviations in the horizontal and vertical directions of less than 2 nm. The latter method is faster, but more sensitive to the contrast and magnification of the video image.

scholarly journals CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic

2019 ◽  
Vol 218 (5) ◽  
pp. 1619-1633 ◽  
Author(s):  
Yuxiao Wang ◽  
Walter Huynh ◽  
Taylor D. Skokan ◽  
Wen Lu ◽  
Arthur Weiss ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Cytoplasmic Dynein ◽  
Receptor Activation ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Motor ◽  
Ef Hands

Cytoplasmic dynein is a minus end–directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated, but not Rab45-mediated, dynein motility is activated by calcium in vitro. In Jurkat T cells, elevation of intracellular calcium activates CRACR2a-mediated dynein transport. We further found that T cell receptor activation induces the formation of CRACR2a puncta at the plasma membrane, which initially associate with the actin cortex and subsequently detach and travel along microtubules, suggestive of an endocytic process. These results provide the first examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a–dynein in calcium-regulated endocytic trafficking.

scholarly journals Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Xiaoxia Ye ◽  
Mingming Zhu ◽  
Xiaohang Che ◽  
Huiyang Wang ◽  
Xing-Jie Liang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Microglial Activation ◽  
Adaptor Protein ◽  
Mtor Pathway ◽  
Microglial Cells ◽  
Inflammatory Factors ◽  
Intraventricular Injection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autophagic Flux

Abstract Background Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. Methods In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 μg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. Conclusions These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

scholarly journals Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0136885 ◽  
Author(s):  
Stéphane Kerbrat ◽  
Benoit Vingert ◽  
Marie-Pierre Junier ◽  
Flavia Castellano ◽  
François Renault-Mihara ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Adaptor Protein

scholarly journals A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

2003 ◽  
Vol 23 (19) ◽  
pp. 6944-6957 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Alexander V. Emelyanov ◽  
Paola Castagnino ◽  
Philip Zegerman ◽  
Andrew J. Bannister ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Adaptor Protein ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Functional Assays ◽  
Two Hybrid ◽  
Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 5961-5971 ◽  
Author(s):  
Fanny Baran-Marszak ◽  
Hajer Magdoud ◽  
Christophe Desterke ◽  
Anabell Alvarado ◽  
Claudine Roger ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Adaptor Protein ◽  
Jak2 V617f ◽  
Negative Control ◽  
Sh2 Domain ◽  
Expression Level ◽  
Activating Mutations ◽  
Thrombopoietin Receptor ◽  
Dependent Cell

Abstract Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

Nontypeable Haemophilus influenzae infection impedes Pseudomonas aeruginosa colonization and persistence in mouse respiratory tract

2021 ◽  
Author(s):  
Natalie Lindgren ◽  
Lea Novak ◽  
Benjamin C. Hunt ◽  
Melissa S. McDaniel ◽  
W. Edward Swords
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Young Patients ◽  
Nontypeable Haemophilus Influenzae ◽  
Potential Significance ◽  
And Diffusion

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa ( Pa ). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

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