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2.
Front Immunol ; 14: 1272055, 2023.
Article in English | MEDLINE | ID: mdl-37942313

ABSTRACT

Conventional type 1 dendritic cells (cDC1s) are superior in antigen cross-presentation and priming CD8+ T cell anti-tumor immunity and thus, are a target of high interest for cancer immunotherapy. Type I interferon (IFN) is a potent inducer of antigen cross-presentation, but, unfortunately, shows only modest results in the clinic given the short half-life and high toxicity of current type I IFN therapies, which limit IFN exposure in the tumor. CD8+ T cell immunity is dependent on IFN signaling in cDC1s and preclinical studies suggest targeting IFN directly to cDC1s may be sufficient to drive anti-tumor immunity. Here, we engineered an anti-XCR1 antibody (Ab) and IFN mutein (IFNmut) fusion protein (XCR1Ab-IFNmut) to determine whether systemic delivery could drive selective and sustained type I IFN signaling in cDC1s leading to anti-tumor activity and, in parallel, reduced systemic toxicity. We found that the XCR1Ab-IFNmut fusion specifically enhanced cDC1 activation in the tumor and spleen compared to an untargeted control IFN. However, multiple treatments with the XCR1Ab-IFNmut fusion resulted in robust anti-drug antibodies (ADA) and loss of drug exposure. Using other cDC1-targeting Ab-IFNmut fusions, we found that localizing IFN directly to cDC1s activates their ability to promote ADA responses, regardless of the cDC1 targeting antigen. The development of ADA remains a major hurdle in immunotherapy drug development and the cellular and molecular mechanisms governing the development of ADA responses in humans is not well understood. Our results reveal a role of cDC1s in ADA generation and highlight the potential ADA challenges with targeting immunostimulatory agents to this cellular compartment.


Subject(s)
Interferon Type I , Neoplasms , Humans , Interferon Type I/metabolism , CD8-Positive T-Lymphocytes , Dendritic Cells , Antigen Presentation
4.
Sci Rep ; 11(1): 3012, 2021 02 04.
Article in English | MEDLINE | ID: mdl-33542328

ABSTRACT

Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.

5.
Cell Rep ; 12(12): 2121-30, 2015 Sep 29.
Article in English | MEDLINE | ID: mdl-26387943

ABSTRACT

Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.


Subject(s)
Adaptor Protein Complex 2/metabolism , Clathrin/metabolism , Endocytosis/genetics , Epithelial Cells/metabolism , Genome, Human , Support Vector Machine , Adaptor Protein Complex 2/genetics , Base Sequence , Cell Line, Tumor , Clathrin/genetics , Dynamin II , Dynamins/genetics , Dynamins/metabolism , Epithelial Cells/cytology , Exons , Female , Humans , Introns , Mammary Glands, Human/cytology , Mammary Glands, Human/metabolism , Molecular Sequence Data
6.
Mol Biol Cell ; 26(8): 1509-22, 2015 Apr 15.
Article in English | MEDLINE | ID: mdl-25694450

ABSTRACT

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.


Subject(s)
Actin Cytoskeleton/metabolism , Clathrin , Endocytosis/physiology , Peptide Hydrolases/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Cell Membrane/metabolism , Microfilament Proteins/metabolism , Myosins/metabolism , Peptide Hydrolases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction
7.
J Cell Sci ; 125(Pt 5): 1329-41, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22303001

ABSTRACT

Podosomes are dynamic actin-based structures that mediate adhesion to the extracellular matrix and localize matrix degradation to facilitate cell motility and invasion. Drebrin-like protein (DBNL), which is homologous to yeast mAbp1 and is therefore known as mammalian actin-binding protein 1 (mAbp1), has been implicated in receptor-mediated endocytosis, vesicle recycling and dorsal ruffle formation. However, it is not known whether mAbp1 regulates podosome formation or cell invasion. In this study, we found that mAbp1 localizes to podosomes and is necessary for the formation of podosome rosettes in Src-transformed fibroblasts. Despite their structural similarity, mAbp1 and cortactin play distinct roles in podosome regulation. Cortactin was necessary for the formation of podosome dots, whereas mAbp1 was necessary for the formation of organized podosome rosettes in Src-transformed cells. We identified specific Src phosphorylation sites, Tyr337 and Tyr347 of mAbp1, which mediate the formation of podosome rosettes and degradation of the ECM. In contrast to dorsal ruffles, the interaction of mAbp1 with WASP-interacting protein (WIP) was not necessary for the formation of podosome rosettes. Finally, we showed that depletion of mAbp1 increased invasive cell migration, suggesting that mAbp1 differentially regulates matrix degradation and cell invasion. Collectively, our findings identify a role for mAbp1 in podosome rosette formation and cell invasion downstream of Src.


Subject(s)
Cell Transformation, Neoplastic , Cell-Matrix Junctions/physiology , Cell-Matrix Junctions/ultrastructure , Microfilament Proteins/metabolism , src-Family Kinases/metabolism , 3T3 Cells , Actin Cytoskeleton , Animals , Carrier Proteins/metabolism , Cell Adhesion , Cell Line , Cell Line, Transformed , Cell Movement , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cortactin/metabolism , Cytoskeletal Proteins , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Fibroblasts/physiology , Membrane Glycoproteins , Mice , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation , Platelet Glycoprotein GPIb-IX Complex , Protein Binding , RNA Interference , RNA, Small Interfering , src Homology Domains
8.
Methods Mol Biol ; 769: 111-36, 2011.
Article in English | MEDLINE | ID: mdl-21748673

ABSTRACT

Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.


Subject(s)
Cell-Matrix Junctions/metabolism , Extracellular Matrix/metabolism , Gelatin/metabolism , Single-Cell Analysis/methods , Cell Differentiation , Cell Line , Chemotaxis , Coated Materials, Biocompatible , Focal Adhesions/metabolism , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Macrophages/cytology , Macrophages/physiology , Microscopy/methods , Recombinant Fusion Proteins/metabolism , Time-Lapse Imaging , Vinculin/biosynthesis , Vinculin/genetics
9.
J Biol Chem ; 286(12): 9998-10006, 2011 Mar 25.
Article in English | MEDLINE | ID: mdl-21270128

ABSTRACT

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.


Subject(s)
Calpain/metabolism , Cell Movement/physiology , Focal Adhesions/metabolism , Paxillin/metabolism , Amino Acid Motifs , Amino Acid Substitution , Calpain/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Focal Adhesions/genetics , Glycoproteins/genetics , Glycoproteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Paxillin/genetics , Point Mutation , Zyxin
11.
Mol Biol Cell ; 21(1): 186-97, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19910490

ABSTRACT

Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor-induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor-induced dorsal ruffle formation through its interaction with WIP.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane Structures/drug effects , Cell Membrane Structures/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Platelet-Derived Growth Factor/pharmacology , Adaptor Proteins, Signal Transducing/chemistry , Animals , Calpain/metabolism , Cortactin/metabolism , Gene Knockdown Techniques , Humans , Mice , Muscle Proteins/chemistry , Mutant Proteins/metabolism , NIH 3T3 Cells , Protein Binding/drug effects , Protein Transport/drug effects , src Homology Domains
12.
J Cell Biol ; 185(2): 357-70, 2009 Apr 20.
Article in English | MEDLINE | ID: mdl-19364917

ABSTRACT

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.


Subject(s)
Breast Neoplasms , Cell Surface Extensions/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/metabolism , Neoplasm Invasiveness , src-Family Kinases/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/physiology , Female , Focal Adhesion Protein-Tyrosine Kinases/genetics , Humans , Paxillin/genetics , Paxillin/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tyrosine/metabolism
13.
Lab Chip ; 8(9): 1507-15, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18818806

ABSTRACT

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.


Subject(s)
Chemotaxis , Animals , Cell Line, Tumor , Models, Biological , Neutrophils/cytology , Nonlinear Dynamics , Rats
14.
Eur J Cell Biol ; 87(8-9): 507-15, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18343530

ABSTRACT

Podosomes and the immune synapse are integrin-mediated adhesive structures that share a common ring-like morphology. Both podosomes and immune synapses have a central core surrounded by a peripheral ring containing talin, vinculin and paxillin. Recent progress suggests significant parallels between the regulatory mechanisms that contribute to the formation of these adhesive structures. In this review, we compare the structures, functions and regulation of podosomes and the immune synapse.


Subject(s)
Cell Surface Extensions/physiology , T-Lymphocytes/immunology , Actins/metabolism , Animals , Antigen-Presenting Cells/immunology , Cell Adhesion , Cell Surface Extensions/ultrastructure , Extracellular Matrix/metabolism , Humans , Integrins/metabolism , Models, Biological , Paxillin/metabolism , Signal Transduction , T-Lymphocytes/ultrastructure , Talin/metabolism , Vinculin/metabolism , rho GTP-Binding Proteins/metabolism
15.
J Cell Biol ; 180(5): 957-71, 2008 Mar 10.
Article in English | MEDLINE | ID: mdl-18332219

ABSTRACT

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.


Subject(s)
Breast Neoplasms/metabolism , Calpain/metabolism , Carcinoma/metabolism , Neoplasm Invasiveness/physiopathology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Breast Neoplasms/physiopathology , CSK Tyrosine-Protein Kinase , Calpain/genetics , Carcinoma/physiopathology , Cell Adhesion/physiology , Cell Line , Cell Line, Tumor , Cell Movement/physiology , Down-Regulation/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Female , Humans , Mutation/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Signal Transduction/physiology , src-Family Kinases
16.
Methods Enzymol ; 426: 47-67, 2007.
Article in English | MEDLINE | ID: mdl-17697879

ABSTRACT

Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration. Furthermore, we outline detailed procedures to examine focal adhesion assembly and disassembly using time-lapse fluorescent microscopy. Finally, we provide methods for the analysis of podosomes, which are highly dynamic adhesive structures that form in immune cells and invasive cancer cells.


Subject(s)
Cell Movement/physiology , Integrins/physiology , Animals , Humans
17.
Arch Biochem Biophys ; 449(1-2): 164-70, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16554018

ABSTRACT

Mannan-binding lectin-associated serine proteases (MASPs) are secreted as single-chain precursors and processed into two disulfide bond-linked chains. MASP-3 and MASP-1, derived from the same gene, contain identical A chains, but entirely different catalytic domain-containing B chains. In contrast to MASP-1 and MASP-2, the proteinase activity of MASP-3 has not been described previously. We show here the proteolytic activity of the purified recombinant human MASP-3 catalytic domain toward peptides and protein substrates. Among the fluorogenic peptides tested, it specifically cleaved peptides with Arg at the P1 position. Among seven insulin-like growth factor-binding proteins, it selectively cleaved IGFBP-5, which is the first protein substrate identified for MASP-3. All three cleavage sites identified contained Arg or Lys at the P1 position and Pro at the P2 position. As compared to MASP-1 and MASP-2, MASP-3 has distinct substrate specificity and inhibitor profile. These results should be useful for further studies of the structure and function of human MASP-3.


Subject(s)
Mannose-Binding Lectins/chemistry , Mannose-Binding Protein-Associated Serine Proteases/chemistry , Peptides/chemistry , Binding Sites , Enzyme Activation , Insulin-Like Growth Factor Binding Protein 5 , Protein Binding , Protein Structure, Tertiary , Substrate Specificity
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