Lissencephaly-1 is a context-dependent regulator of the human dynein complex

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

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Dynactin Is Required for Microtubule Anchoring at Centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.147.2.321 ◽

1999 ◽

Vol 147 (2) ◽

pp. 321-334 ◽

Cited By ~ 247

Author(s):

N.J. Quintyne ◽

S.R. Gill ◽

D.M. Eckley ◽

C.L. Crego ◽

D.A. Compton ◽

...

Keyword(s):

Mitotic Cell ◽

Cytoplasmic Dynein ◽

Microtubule Organization ◽

Cultured Fibroblasts ◽

Cell Extracts ◽

Dynein Motor ◽

Mitotic Spindle Assembly ◽

Dynactin Complex

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

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Dynein-Mediated Cargo Transport in Vivo

The Journal of Cell Biology ◽

10.1083/jcb.148.5.945 ◽

2000 ◽

Vol 148 (5) ◽

pp. 945-956 ◽

Cited By ~ 160

Author(s):

Steven P. Gross ◽

Michael A. Welte ◽

Steven M. Block ◽

Eric F. Wieschaus

Keyword(s):

Lipid Droplets ◽

Developmental Stages ◽

Cytoplasmic Dynein ◽

Chain Gene ◽

Unit Distance ◽

Cargo Transport ◽

Single Droplets ◽

Drosophila Embryos

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

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Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽

10.1182/blood.v78.9.2283.bloodjournal7892283 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2283-2290 ◽

Cited By ~ 1

Author(s):

H Hoogendoorn ◽

CH Toh ◽

ME Nesheim ◽

AR Giles

Keyword(s):

Complex Formation ◽

Active Site ◽

Metal Ion ◽

Protein C ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

High Molecular Weight Complex

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

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Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1769 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1769-1777 ◽

Cited By ~ 140

Author(s):

X Wang ◽

M Kiledjian ◽

I M Weiss ◽

S A Liebhaber

Keyword(s):

Complex Formation ◽

Mrna Stability ◽

Untranslated Region ◽

Binding Protein ◽

Erythroid Cell ◽

Globin Mrna ◽

Rnp Complex ◽

Alpha Globin

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.

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Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽

10.1182/blood.v78.9.2283.2283 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2283-2290 ◽

Cited By ~ 27

Author(s):

H Hoogendoorn ◽

CH Toh ◽

ME Nesheim ◽

AR Giles

Keyword(s):

Complex Formation ◽

Active Site ◽

Metal Ion ◽

Protein C ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

High Molecular Weight Complex

Abstract In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

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Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

The Journal of Cell Biology ◽

10.1083/jcb.131.2.411 ◽

1995 ◽

Vol 131 (2) ◽

pp. 411-425 ◽

Cited By ~ 89

Author(s):

M McGrail ◽

J Gepner ◽

A Silvanovich ◽

S Ludmann ◽

M Serr ◽

...

Keyword(s):

Temporal Distribution ◽

Cytoplasmic Dynein ◽

Chain Gene ◽

Gene Products ◽

Wild Type ◽

Microtubule Associated Proteins ◽

Dynein Motor ◽

Associated Proteins

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

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Isolation and Properties of the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL

Journal of Bacteriology ◽

10.1128/jb.181.15.4461-4468.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4461-4468 ◽

Cited By ~ 44

Author(s):

Tracy Money ◽

Tamera Jones ◽

Ray Dixon ◽

Sara Austin

Keyword(s):

Complex Formation ◽

Azotobacter Vinelandii ◽

Binding Protein ◽

Protein Protein Interaction ◽

Amino Terminal ◽

Cell Extracts ◽

Enhancer Binding Protein ◽

Adenosine Nucleotides

ABSTRACT In Azotobacter vinelandii, activation ofnif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.

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A severely defective TATA-binding protein-TFIIB interaction does not preclude transcriptional activation in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1336 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1336-1345 ◽

Cited By ~ 26

Author(s):

M Lee ◽

K Struhl

Keyword(s):

Dna Binding ◽

Complex Formation ◽

Transcriptional Activation ◽

Binding Protein ◽

Tata Binding Protein ◽

Yeast Cells ◽

Synthetic Lethal ◽

Binding Surface

In yeast cells, mutations in the TATA-binding protein (TBP) that disrupt the interaction with the TATA element or with TFIIA can selectively impair the response to acidic activator proteins. We analyzed the transcriptional properties of TBP derivatives in which residues that directly interact with TFIIB were replaced by alanines. Surprisingly, a derivative with a 50-fold defect in TBP-TFIIB-TATA complex formation in vitro (E188A) supports viability and responds efficiently to activators in vivo. The E186A derivative, which displays a 100-fold defect in TBP-TFIIB-TATA complex formation, does not support viability, yet it does respond to activators. Conversely, the L189A mutation, which has the mildest effect on the interaction with TFIIB (10-fold), can abolish transcriptional activation and cell viability when combined with mutations on the DNA-binding surface. This "synthetic lethal" effect is not observed with E188A, suggesting that the previously described role of L189 in transcriptional activation may be related to its location on the DNA-binding surface and not to its interaction with TFIIB. Finally, when using TBP mutants defective on multiple interaction surfaces, we observed synthetic lethal effects between mutations on the TFIIA and TFIIB interfaces but found that mutations implicated in association with polymerase II and TFIIF did not have significant effects in vivo. Taken together, these results argue that, unlike the TBP-TATA and TBP-TFIIA interactions, the TBP-TFIIB interaction is not generally limiting for transcriptional activation in vivo.

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C4b-binding protein α-chain enhances antitumor immunity by facilitating the accumulation of tumor-infiltrating lymphocytes in the tumor microenvironment in pancreatic cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02019-0 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Kosuke Sasaki ◽

Shigetsugu Takano ◽

Satoshi Tomizawa ◽

Yoji Miyahara ◽

Katsunori Furukawa ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Binding Protein ◽

Combined Treatment ◽

Tumor Infiltrating Lymphocytes ◽

Pdac Cell ◽

Α Chain ◽

Infiltrating Lymphocytes

Abstract Background Recent studies indicate that complement plays pivotal roles in promoting or suppressing cancer progression. We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). However, its mechanism of action remains unclear. Here, we elucidated the functional roles of C4BPA in PDAC cells and the tumor microenvironment. Methods We assessed stromal C4BPA, the C4BPA binding partner CD40, and the number of CD8+ tumor-infiltrating lymphocytes in resected human PDAC tissues via immunohistochemical staining. The biological functions of C4BPA were investigated in peripheral blood mononuclear cells (PBMCs) and human PDAC cell lines. Mouse C4BPA (mC4BPA) peptide, which is composed of 30 amino acids from the C-terminus and binds to CD40, was designed for further in vitro and in vivo experiments. In a preclinical experiment, we assessed the efficacy of gemcitabine plus nab-paclitaxel (GnP), dual immune checkpoint blockades (ICBs), and mC4BPA peptide in a mouse orthotopic transplantation model. Results Immunohistochemical analysis revealed that high stromal C4BPA and CD40 was associated with favorable PDAC prognosis (P=0.0005). Stromal C4BPA strongly correlated with the number of CD8+ tumor-infiltrating lymphocytes (P=0.001). In in vitro experiments, flow cytometry revealed that recombinant human C4BPA (rhC4BPA) stimulation increased CD4+ and CD8+ T cell numbers in PBMCs. rhC4BPA also promoted the proliferation of CD40-expressing PDAC cells. By contrast, combined treatment with gemcitabine and rhC4BPA increased PDAC cell apoptosis rate. mC4BPA peptide increased the number of murine T lymphocytes in vitro and the number of CD8+ tumor-infiltrating lymphocytes surrounding PDAC tumors in vivo. In a preclinical study, GnP/ICBs/mC4BPA peptide treatment, but not GnP treatment, led to the accumulation of a greater number of CD8+ T cells in the periphery of PDAC tumors and to greater tumor regression than did control treatment. Conclusions These findings demonstrate that the combination of GnP therapy with C4BPA inhibits PDAC progression by promoting antitumor T cell accumulation in the tumor microenvironment.

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Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽

10.1182/blood.v83.9.2516.2516 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2516-2525 ◽

Cited By ~ 13

Author(s):

K Meszaros ◽

S Aberle ◽

R Dedrick ◽

R Machovich ◽

A Horwitz ◽

...

Keyword(s):

Tissue Factor ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Binding Protein ◽

Mononuclear Phagocytes ◽

Factor Vii ◽

Peripheral Blood Mononuclear ◽

Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

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