ABSTRACT
The mechanism of cargo coupling to kinesin motor proteins is a fundamental issue in organelle transport along microtubules. Kinectin has been postulated to function as a membrane anchor protein that attaches various organelles to the prototype motor protein kinesin. To verify the biological relevance of kinectin in vivo, the murine kinectin gene was disrupted by homologous recombination. Unexpectedly, kinectin-deficient mice were viable and fertile, and no gross abnormalities were observed up to 1 year of age. The assembly of the endoplasmic reticulum was essentially unaffected in kinectin-deficient cells. Mitochondria appeared to be correctly distributed throughout the cytoplasm along the microtubules. Furthermore, the stationary distribution and the bidirectional movement of lysosomes did not depend on kinectin. Kinectin-deficient phagocytes internalized and cleared bacteria, indicating that phagosome trafficking and maturation are functional without kinectin. Thus, these data unequivocally indicate that kinectin is not essential for trafficking of lysosomes, phagosomes, and mitochondria in vivo.
Subject(s)
Blood Proteins/physiology , Lysosomes/metabolism , Membrane Proteins , Phagosomes/physiology , Alleles , Animals , Biological Transport , Blood Proteins/genetics , Cell Line , Humans , Intracellular Membranes/metabolism , Mice , Mice, Knockout , Mutagenesis , Organelles/metabolism , Phagocytosis , Phagosomes/metabolism , Subcellular FractionsABSTRACT
The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.
Subject(s)
Epidermis/physiology , Homeostasis , Proteins/genetics , Sphingomyelin Phosphodiesterase/metabolism , Amino Acid Sequence , Animals , Cytotoxicity, Immunologic , Enzyme Activation/drug effects , Epidermis/injuries , Gene Targeting , Intracellular Signaling Peptides and Proteins , Killer Cells, Natural , Mice , Mice, Mutant Strains , Permeability , Sequence Homology, Amino Acid , Signal Transduction , Thymus Gland/cytology , Thymus Gland/immunology , Tumor Necrosis Factor-alpha/pharmacology , Vesicular Transport Proteins , Wound HealingABSTRACT
The biological functions mediated by the death domain of the 55-kDa tumor necrosis factor receptor (TNFRp55) in vivo are still elusive. TNFRp55 mutants lacking a functional death domain were expressed in TNFRp55-/- and in TNFRp55+/- mice as transgenes under control of the H-2Kb promoter. Analysis of these animals revealed that signals originating from the TNFRp55 death domain are indispensable for the protection against Listeria monocytogenes, the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the spleen and the development of splenic germinal centers. Furthermore, the transgenic coexpression of the TNFRp55 mutants in TNFRp55+/- mice exerts a dominant negative effect on the signaling of the endogenous receptor chains in vivo.
Subject(s)
Antigens, CD/immunology , Germinal Center/immunology , Listeriosis/immunology , Receptors, Tumor Necrosis Factor/immunology , Animals , Antigens, CD/genetics , Immunity, Innate/genetics , Mice , Mice, Transgenic , Mutation , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Tumor Necrosis Factor-alpha/immunologyABSTRACT
The formation of germinal centers (GCs) represents a crucial step in the humoral immune response. Recent studies using gene-targeted mice have revealed that the cytokines tumor necrosis factor (TNF), lymphotoxin (LT) alpha, and LTbeta, as well as their receptors TNF receptor p55 (TNFRp55) and LTbetaR play essential roles in the development of GCs. To establish in which cell types expression of LTbetaR, LTbeta, and TNF is required for GC formation, LTbetaR-/-, LTbeta-/-, TNF-/-, B cell-deficient (BCR-/-), and wild-type mice were used to generate reciprocal or mixed bone marrow (BM) chimeric mice. GCs, herein defined as peanut agglutinin-binding (PNA+) clusters of centroblasts/centrocytes in association with follicular dendritic cell (FDC) networks, were not detectable in LTbetaR-/- hosts after transfer of wild-type BM. In contrast, the GC reaction was restored in LTbeta-/- hosts reconstituted with either wild-type or LTbetaR-/- BM. In BCR-/- recipients reconstituted with compound LTbeta-/-/BCR-/- or TNF-/-/BCR-/- BM grafts, PNA+ cell clusters formed in splenic follicles, but associated FDC networks were strongly reduced or absent. Thus, development of splenic FDC networks depends on expression of LTbeta and TNF by B lymphocytes and LTbetaR by radioresistant stromal cells.
