RESUMEN
BACKGROUND: Some of the mechanisms underlying cell division and partitioning of the cellular components into the daughter cells are well known. Within the endomembrane system, there is a general cessation of membrane traffic, including endocytosis and endosome fusion, at the onset of mitosis. However, the fate of endosomes and lysosomes during mitosis has been less well studied. RESULTS: Using video and confocal microscopy of living cells, we show here that endosomes and lysosomes remain intact and separate during mitosis. The segregation into daughter cells takes place by coordinated movements, and during cytokinesis, these organelles accumulate in the vicinity of the microtubule organization center. However, partitioning into daughter cells is not more accurate than a calculated stochastic distribution, despite the apparent order to the process. CONCLUSION: We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.
Asunto(s)
Endosomas , Lisosomas , Mitosis , Animales , Línea Celular , Perros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Transporte VesicularRESUMEN
The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Transducción de Señal/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/análisis , Membrana Celular/química , Membrana Celular/inmunología , Pollos , Clonación Molecular , ADN Complementario , Endosomas/química , Endosomas/inmunología , Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Leucina , Mamíferos , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
CD1d is a member of the CD1 polypeptide family that represents a new arm of host defense against invading pathogens. In our previous work (Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P., and Bakke, O. (1999) J. Immunol. 162, 1488-1495) we have shown that CD1d contained a classic tyrosine-based internalization signal (YQGV) in its short cytoplasmic tail. CD1d is expressed in polarized epithelial cells, and we found that the cytoplasmic tail of CD1d also contained information for basolateral sorting. Interestingly, a mutation of the critical tyrosine residue of the endosomal sorting signal did not result in the loss of basolateral targeting of the mutant CD1d. To search for a basolateral sorting signal we have constructed a full set of alanine mutants, but no single alanine substitution inactivated the signal. However, deletions or mutations of either the C-terminal valine/leucine pair or the critical tyrosine residue from the internalization signal and either residue from the C-terminal valine/leucine pair inactivated basolateral sorting. Our data thus suggest that the cytoplasmic tail contains two overlapping basolateral signals, one tyrosine- and the other leucine-based, each being sufficient to direct CD1d to the basolateral membrane of polarized Madin-Darby canine kidney cells.
Asunto(s)
Antígenos CD1/metabolismo , Polaridad Celular , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Animales , Antígenos CD1d , Transporte Biológico , Compartimento Celular , Perros , Endosomas/metabolismo , Riñón/citología , Datos de Secuencia MolecularRESUMEN
MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the beta-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/fisiología , Polaridad Celular/inmunología , Citoplasma/inmunología , Antígeno HLA-DR1/metabolismo , Antígenos de Histocompatibilidad Clase II/fisiología , Leucina/fisiología , Fragmentos de Péptidos/fisiología , Transducción de Señal/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Transporte Biológico/inmunología , Compartimento Celular/genética , Compartimento Celular/inmunología , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Perros , Endosomas/inmunología , Endosomas/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Humanos , Riñón , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , TransfecciónRESUMEN
Protection against desiccation-induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by-products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation-induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free-radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol-requiring anti-oxidants with one conserved cysteine residue (1-Cys), and displays in vitro anti-oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy-related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non-dormant seeds. Our data identify the location of this seed-specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C-terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.
Asunto(s)
Núcleo Celular/química , Hordeum/química , Peroxidasas , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Hordeum/citología , Hordeum/embriología , Datos de Secuencia Molecular , Señales de Localización Nuclear , Peroxirredoxinas , Proteínas de Plantas/química , Semillas/química , Fracciones Subcelulares/metabolismoRESUMEN
Targeting of many transmembrane proteins to post-Golgi compartments is dependent on cytoplasmically exposed sorting signals. The most widely used signals conform to the tyrosine- or the leucine-based motifs. Both types of signals have been implicated in protein localization to the same intracellular compartments, but previous results from both cell-free experiments and studies of transfected cell lines have indicated that the two types of signals interact with separate components of the sorting machinery. We have overexpressed several transmembrane proteins in stably transfected Madin-Darby canine kidney cells using an inducible promoter system. Overexpression of proteins containing tyrosine- or leucine-based sorting signals resulted in reduced internalization of the transferrin receptor, whereas recycling and polarized distribution was not influenced. Our results indicate that proteins with tyrosine- and leucine-based sorting signals can be transported along common saturable pathways.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Receptores de Transferrina/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Línea Celular , Citoplasma/metabolismo , Perros , Aparato de Golgi/metabolismo , Leucina , Proteínas de la Membrana/genética , Ratones , Regiones Promotoras Genéticas/genética , Receptores de Transferrina/genética , Transducción de Señal/genética , Transfección , TirosinaRESUMEN
The CD1 family of polypeptides is divided into two groups, the CD1b and CD1d group. Both groups are involved in stimulation of T cell response. Molecules of the CD1b group can present Ag derived from bacterial cell walls to T cells; the process of Ag acquisition is thought to take place in endosomes. Little is known about Ag presentation by CD1d. We therefore studied the intracellular trafficking of human CD1d in Madin-Darby canine kidney (MDCK) and COS cells. CD1d was found in endosomal compartments after its internalization from the plasma membrane. It is therefore possible that CD1d acquires its yet unidentified exogenous ligand in the same compartments as the MHC class II and CD1b molecules. CD1d contains a tyrosine-based sorting signal in its cytoplasmic tail that is necessary for internalization. Furthermore, the cytoplasmic tail of CD1d also contains a signal for basolateral sorting that is, however, different from the internalization signal.
Asunto(s)
Antígenos CD1/química , Antígenos CD1/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos CD1/genética , Células COS , Línea Celular , Polaridad Celular , Citoplasma/inmunología , Perros , Endocitosis , Humanos , Microscopía Electrónica , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Tirosina/químicaRESUMEN
In this review we focus on the traffic of MHC class II and endocytosed antigens to intracellular compartments where antigenic peptides are loaded. We also discuss briefly the nature of the peptide loading compartment and the sorting signals known to direct antigen receptors and MHC class II and associated molecules to this location. MHC class II molecules are expressed on a variety of polarized epithelial and endothelial cells, and polarized cells are thus potentially important for antigen presentation. Here we review some cell biological aspects of polarized sorting of MHC class II and the associated invariant chain and the signals that are involved in the sorting process to the basolateral domain. The molecules involved in sorting and loading of peptide may modulate antigen presentation, and in particular we discuss how invariant chain may change the cellular phenotype and the kinetics of the endosomal pathway.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Transporte Biológico Activo , Compartimento Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Polaridad Celular , Endosomas/inmunología , Endosomas/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Humanos , Modelos Moleculares , Péptidos/genética , Conformación Proteica , Transducción de SeñalRESUMEN
The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains signals for transport to endocytic compartments where the class II molecules bind antigenic peptides for presentation to CD4+ T cells. Two leucine-based signals in the Ii cytoplasmic tail can be independently recognized for endosomal sorting of Ii, and we have recently shown that each signal is sufficient for basolateral sorting and internalization of Ii in polarized Madine Darby Canine Kidney (MDCK) II cells. The recognition motif for endosomal sorting is complex and consists of two critical leucine-like residues as well as surrounding amino acids. Here, we have analyzed the importance of residues surrounding the membrane-distal leucine-based signal in basolateral sorting and internalization of Ii in MDCK II cells. We find that the DDQxxLI motif is involved in both sorting events indicating the presence of similar signal recognition components both at the TGN and at the plasma membrane. The identical motif is required for endosomal localization and internalization of Ii also in simian COS cells and the human HeLa and M1 cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Leucina/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Sitios de Unión , Células COS , Línea Celular , Polaridad Celular , Perros , Células HeLa , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Líquido Intracelular , Datos de Secuencia Molecular , Mutagénesis , Serina/metabolismoRESUMEN
Transport of polymeric Igs (pIgA and pIgM) across secretory epithelia is mediated by the polymeric Ig receptor (pIgR), also known as the transmembrane secretory component. Compared with local production, external transfer of pIgA is favored 6- to 12-fold over that of pIgM on a molar basis. This transfer may be modulated at several levels: diffusion through matrix and basement membranes, ligand affinity for pIgR, and efficiency of epithelial transcytosis. To investigate these possibilities, we compared the ability of Madin-Darby canine kidney epithelial cells transfected with human pIgR to transport pIgA vs pIgM from the basolateral to the apical face, and examined the inhibitory effect of various filter types used for mounting of the monolayer. Binding data showed that pIgR bound pIgA and pIgM with similar affinity. Internalization of both ligands was fast and took place at similar rates; transcytosis was also found to be equally efficient at the molar level. Thus, the overall rate of transport across the epithelial monolayer was comparable for pIgA and pIgM, and was not further enhanced by ligand stimulation over a 20-fold increased concentration level. Conversely, pIgA had a considerable advantage over pIgM in passive diffusion assays performed in vitro. Moreover, in situ immunofluorescence staining showed retention of IgM over IgA and IgG in mucosal basement membrane zones, in contrast to the preferential epithelial uptake of IgA and, less so, IgM. The biologic consequences of the highly efficient epithelial pIg transport, and the diffusion advantage of pIgA over pIgM, are discussed in relation to the evolution and function of secretory Abs.
Asunto(s)
Inmunoglobulina A/metabolismo , Inmunoglobulina M/metabolismo , Riñón/inmunología , Receptores Inmunológicos/inmunología , Animales , Transporte Biológico/inmunología , Línea Celular , Dimerización , Perros , Epitelio/inmunología , Epitelio/metabolismo , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina M/inmunología , Riñón/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
Foreign antigens are internalized by antigen presenting cells by endocytosis and processed to peptides. To enable presentation of antigenic peptides by MHC class II molecules, these molecules have to be sorted to endosomal compartments where they can meet and bind the peptides. Invariant chain is complexed with MHC class II molecules and contains sorting signals responsible for MHC class II accumulation in endosomes. Invariant chain also has several other features contributing to the immune system's specific combat against invaders.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Diferenciación de Linfocitos B , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Transporte Biológico/inmunología , Humanos , Transducción de Señal/inmunologíaRESUMEN
We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (delta 15Ii) or 23 (delta 23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas delta 15Ii did not affect antigen presentation by class II molecules, delta 23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of delta 23Ii can be internalized, followed by degradation of delta 23Ii and return of free class II alpha beta heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.