Biomedical subjects

S Kaech

Publications and source records attributed to S Kaech.

18 recordsLinked to original sources

PubMed2005

Differentiation of CD8 T cells in response to acute and chronic viral infections: implications for HIV vaccine development.

Successful HIV vaccine strategies will likely require the induction of robust cellular immune responses, in addition to strong humoral responses. Unfortunately, there is no clear molecular definition of an effective HIV-specific CD8 T cell response. In this review, we discuss the differentiation of CD8 T cells in response to acute and chronic viral infections. We then apply concepts derived from these studies to predict the desirable characteristics of HIV-specific CD8 T cell memory.

AIDS Vaccines↗

PubMed2001

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture.

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Animals↗

PubMed2001

Cytoskeletal microdifferentiation: a mechanism for organizing morphological plasticity in dendrites.

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubule-associated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Actins↗

PubMed2000

Glutamate receptors regulate actin-based plasticity in dendritic spines.

Dendritic spines at excitatory synapses undergo rapid, actin-dependent shape changes which may contribute to plasticity in brain circuits. Here we show that actin dynamics in spines are potently inhibited by activation of either AMPA or NMDA subtype glutamate receptors. Activation of either receptor type inhibited actin-based protrusive activity from the spine head. This blockade of motility caused spines to round up so that spine morphology became both more stable and more regular. Inhibition of spine motility by AMPA receptors was dependent on postsynaptic membrane depolarization and influx of Ca 2+ through voltage-activated channels. In combination with previous studies, our results suggest a two-step process in which spines initially formed in response to NMDA receptor activation are subsequently stabilized by AMPA receptors.

Actins↗

PubMed1999

Volatile anesthetics block actin-based motility in dendritic spines.

Dendritic spines form the postsynaptic contact sites for most excitatory synapses in the brain. Spines occur in a wide range of different shapes that can vary depending on an animal's experience or behavioral status. Recently we showed that spines on living neurons can change shape within seconds in a process that depends on actin polymerization. We have now found that this morphological plasticity is blocked by inhalational anesthetics at concentrations at which they are clinically effective. These volatile compounds also block actin-based motility in fibroblasts, indicating that their action is independent of neuron-specific components and thus identifying the actin cytoskeleton as a general cellular target of anesthetic action. These observations imply that inhibition of actin dynamics at brain synapses occurs during general anesthesia and that inhalational anesthetics are capable of influencing the morphological plasticity of excitatory synapses in the brain.

Actins↗

PubMed1999

Lithium reduces tau phosphorylation: effects in living cells and in neurons at therapeutic concentrations.

BACKGROUND: The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. METHODS: Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. RESULTS: Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concentrations did not alter neuronal morphology. Green fluorescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. CONCLUSIONS: Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders.

Animals↗

PubMed1999

Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.

Actins↗

PubMed1998

Rapid actin-based plasticity in dendritic spines.

Dendritic spines have been proposed as primary sites of synaptic plasticity in the brain. Consistent with this hypothesis, spines contain high concentrations of actin, suggesting that they might be motile. To investigate this possibility, we made video recordings from hippocampal neurons expressing actin tagged with green fluorescent protein (GFP-actin). This reagent incorporates into actin-containing structures and allows the visualization of actin dynamics in living neurons. In mature neurons, recordings of GFP fluorescence revealed large actin-dependent changes in dendritic spine shape, similar to those inferred from previous studies using fixed tissues. Visible changes occurred within seconds, suggesting that anatomical plasticity at synapses can be extremely rapid. As well as providing a molecular basis for structural plasticity, the presence of motile actin in dendritic spines implicates the postsynaptic element as a primary site of this phenomenon.

Actins↗

PubMed1997

Isoform specificity in the relationship of actin to dendritic spines.

Dendritic spines contain high concentrations of actin, but neither the isoforms involved nor the mechanism of accumulation is known. In situ hybridization with specific probes established that beta- and gamma-cytoplasmic actins are selectively expressed at high levels by spine-bearing neurons. Transfecting cultured hippocampal neurons with epitope-tagged actin isoforms showed that cytoplasmic beta- and gamma-cytoplasmic actins are correctly targeted to spines, whereas alpha-cardiac muscle actin, which is normally absent from neurons, formed aggregates in dendrites. The transfected actin cDNAs contained only coding domains, suggesting that spine targeting involves amino acid sequences in the proteins, an interpretation supported by experiments with chimeric cDNAs in which C-terminal actin sequences were found to be determinative in spine targeting. By contrast to actin, microtubule components, including tubulin and MAP2, were restricted to the dendritic shaft domain. The close association of cytoplasmic actins with spines together with their general involvement in cell surface motility further supports the idea that actin motility-based changes in spine shape may contribute to synaptic plasticity.

Actins↗

PubMed1996

Improved lipid-mediated gene transfer into primary cultures of hippocampal neurons.

We have examined lipids as transfection agents to introduce recombinant plasmids into primary cultures of rat hippocampal neurons. By modifying the protocol for transfection mediated by the commercial reagent DOTAP, we were able to achieve a transfection efficiency of about 3%. Expression of various transfected gene products was sustained for several weeks in culture, the neurons developed normally and the transfected gene products were targeted to the appropriate subcellular compartment.

Animals↗

PubMed1996

Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins.

We describe the construction of pBact-NGFP and pBact-CGFP, two expression vectors that incorporate green fluorescent protein (GFP) as a fluorescent tag at the N- or C terminus of the produced protein. When transfected into recipient cells, GFP-tagged proteins can be visualised in the living cells using standard fluorescence microscopy techniques. Using these expression vectors, we have produced GFP-tagged versions of the neuronal microtubule-associated proteins (MAP), MAP2c and Tau34, in a number of different cell types. Both GFP-MAP2c and GFP-Tau34 were fluorescent and retained their ability to bind to microtubules. The pBact-NGFP and pBact-CGFP expression vectors represent a fast and convenient way to produce fluorescently tagged polypeptides of selected sequences encoding whole proteins or fragments for the analysis of function and dynamic events in living cells.

Animals↗

PubMed1996

Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins.

MAP2 and tau are the two most prominent neuron-specific microtubule-associated proteins. They have been implicated in the stabilization of microtubules and consequently of neurite morphology. To investigate their influence on microtubule dynamics, we have tagged both proteins with green fluorescent protein and expressed them in non-neuronal cells. Time-lapse recordings of living cells showed that MAP2 and tau did not significantly affect the rates of microtubule growth and shrinkage. Longer recordings revealed the growth and disappearance of MAP-induced microtubule bundles coinciding with changes in cell shape. This supports the idea that microtubule dynamics are influenced by the cortical cytoskeleton. The dynamics-preserving stabilization of microtubules by MAP2 and tau thus provides a molecular basis for the morphological plasticity reported to exist in established neurites.

Animals↗

PubMed1995

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

Amino Acid Sequence↗

PubMed1993

Myristylation and amino-terminal phosphorylation are required for activation of pp60c-src during mitosis.

pp60c-src, a cellular tyrosine kinase homologous to the retroviral v-src oncogene, becomes transiently activated during mitosis. Activation is accompanied by phosphorylation of three sites in the amino-terminal regulatory domain of the protein, threonine 34, threonine 46 and serine 72. These sites can be phosphorylated in vitro by a cell cycle-regulated kinase, p34cdc2, yet this does not result in increased kinase activity of pp60c-src. pp60c-src is negatively regulated by phosphorylation at tyrosine 527, and it has been shown that this site is transiently dephosphorylated in mitotic cells. The importance of tyrosine 527 in the regulation of pp60c-src is also emphasized by the fact that oncogenic mutants of pp60src lacking tyrosine 527 are constitutively active during the entire cell cycle. Here we report that a non-myristylated mutant of pp60c-src is not activated and only partially phosphorylated at the amino terminus in mitotic cells. Additional mutants lacking one (TTAc-src), two (AASc-src) and three (AAAc-src) cdc2 phosphorylation sites had slightly higher kinase activity than wild-type pp60c-src in interphase cells and were not activated during mitosis. However, all four mutant proteins were still transiently dephosphorylated at tyrosine 527 during mitosis, suggesting that myristylation and amino-terminal phosphorylation may be necessary but are clearly not sufficient for mitosis-specific activation.

3T3 Cells↗

PubMed1991

Association of p60c-src with polyoma virus middle-T antigen abrogating mitosis-specific activation.

Polyoma middle-T antigen is required for tumorigenesis in animals and for viral transformation of a variety of cells in culture (reviewed in ref. 1). Middle-T associates with and thereby activates p60c-src, a cellular tyrosine kinase homologous to the oncogene product of Rous sarcoma virus. Activation of p60c-src by middle-T is accompanied both by dephosphorylation of tyrosine 527, a site which negatively regulates src kinase src kinase activity (reviewed in refs 4-6) and by autophosphorylation on tyrosine 416 (refs 7-10). Phosphoprotein p60c-src is subject to cell cycle-specific regulation. It is most active during mitosis and repressed in interphase. Here we report that mitotic p60c-src is dephosphorylated at tyrosine 527. We also show that in cells expressing middle-T, src kinase activity is high both in mitosis and during interphase. An oncogenic mutant src protein, p60c-src(527F), where tyrosine 527 is substituted by phenylalanine, is also highly active in all phases of the cell cycle.

Animals↗

PubMed1990

Myristylation of pp60c-src is not required for complex formation with polyomavirus middle-T antigen.

Middle-T antigen (middle-T), the transforming gene product of polyomavirus, associates with several cellular tyrosine kinases, such as pp60c-src. Complex formation leads to kinase activation and is essential for cell transformation. Middle-T-associated as well as uncomplexed pp60c-src is predominantly found in the plasma membrane. We transfected mouse 3T3 fibroblasts with a mutated c-src gene (2Ac-src), allowing the expression of a protein containing alanine instead of glycine in position 2 of the primary translation product. Contrary to the wild-type c-src gene product, pp60c-src(2A) was not myristylated and accumulated in the cytoplasm instead of being transferred to cellular membranes. The mutant protein was able to associate with middle-T and was activated similarly to the wild-type c-src gene product. Both wild-type and 2A mutant protein were membrane associated upon complex formation with middle-T. This finding suggests that the putative carboxy-terminal membrane anchor sequence of middle-T is sufficient to hold middle-T-associated pp60c-src(2A) in the plasma membrane.

Animals↗

PubMed1989

Stimulation of pp60c-src kinase activity in FDC-P1 cells by polyoma middle-T antigen and hematopoietic growth factors.

Either of two hematopoietic growth factors, GM-CSF or IL-3, are required for the growth of the bone marrow-derived FDC-P1 cell line. These factors induce cellular tyrosine-specific protein kinases when added to resting cells. The receptors for these factors have not been unambiguously shown to contain a kinase domain. To determine whether src-related kinases, in particular pp60-c-src, are regulated by GM-CSF or IL-3, FDC-P1 cells were transfected with plasmids carrying polyoma middle-T antigen, a potent activator of pp60c-src. Middle-T-expressing cells showed a reduced requirement for GM-CSF and IL-3 and selected clones grew in the absence of these factors. Middle-T formed a complex with pp60c-src and stimulated its in vitro kinase activity 20-50 fold. pp60c-src kinase activity was further increased if middle-T-expressing, factor-independent cells were treated with GM-CSF or IL-3.

Animals↗