Mary N. Teruel

CaMKIIβ Functions As an F-Actin Targeting Module that Localizes CaMKIIα/β Heterooligomers to Dendritic Spines

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase that regulates long-term potentiation and other forms of neuronal plasticity. Functional differences between the neuronal CaMKIIalpha and CaMKIIbeta isoforms are not yet known. Here, we use green fluorescent protein-tagged (GFP-tagged) CaMKII isoforms and show that CaMKIIbeta is bound to F-actin in dendritic spines and cell cortex while CaMKIIalpha is largely a cytosolic enzyme. When expressed together, the two isoforms form large heterooligomers, and a small fraction of CaMKIIbeta is sufficient to dock the predominant CaMKIIalpha to the actin cytoskeleton. Thus, CaMKIIbeta functions as a targeting module that localizes a much larger number of CaMKIIalpha isozymes to synaptic and cytoskeletal sites of action.

Molecular memory by reversible translocation of calcium/calmodulin-dependent protein kinase II.

Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIα (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.

Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction.

(1) The diffusion profile of a protein that originates in a point source can be described as (Crank, J., The Mathematics of Diffusion, Clarendon, Oxford, 2nd ed., 1975): . If the question is how far a protein will get on average away from its origin, the problem can be reduced to integration of 2 · x · F(x,t) from 0 to infinity. This calculation uses the mirror principle of diffusion described in the same reference. The result was used in the equation in the text. (2) The calculation of the range of action of a continuously but locally produced active protein can be obtained by multiplying the probability that a protein will be inactivated at a time t: 1/τ · exp(−t/τ), with the average diffusion distance of the protein in the same time: (4 · D · t/π)1/2 and by calculating the integral of the product over time from 0 to infinity. This calculation was made for two and three dimensions for the equations that were included in the text.*E-mail: tobiasmeyer@stanford.edu, mteruel@stanford.edu

A versatile microporation technique for the transfection of cultured CNS neurons

The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simple and efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique that efficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Microporation allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higher percentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higher levels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporated neurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability to produce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein (GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum. The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherent neurons in a 3 mm2 surface area while requiring as little as 1 microl of the material to be introduced. We conclude that the microporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.

Control of astrocyte Ca2+ oscillations and waves by oscillating translocation and activation of protein kinase C

Abstract Background: Glutamate-induced Ca 2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca 2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca 2+ oscillations and waves. Results: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCγ plasma membrane translocation events, induced rapid oscillations in GFP-PKCγ translocation, and generated GFP-PKCγ translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca 2+ -sensitive C2 domains of PKCγ and were driven by localized Ca 2+ spikes, by oscillations in Ca 2+ concentration, and by propagating Ca 2+ waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCγ or PKCδ that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca 2+ and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca 2+ spikes, suggesting that PKC activation is involved in terminating individual Ca 2+ spikes and waves and in defining the time period between Ca 2+ spikes. Conclusions: Our study suggests that cPKCs have a negative feedback role on Ca 2+ oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca 2+ concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca 2+ spikes and waves and can define the Ca 2+ spike and wave frequencies.

Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells

Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.

Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide

Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression.

Differential codes for free Ca2+–calmodulin signals in nucleus and cytosol

BACKGROUND Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.

Fluorescence imaging of signaling networks

Receptor-triggered signaling processes exhibit complex cross-talk and feedback interactions, with many signaling proteins and second messengers acting locally within the cell. The flow of information in this input-output system can only be understood by tracking where and when local signaling activities are induced. Systematic strategies are therefore needed to measure the localization and translocation of all signaling proteins, and to develop fluorescent biosensors that can track local signaling activities in individual cells. Such a biosensor tool chest can be based on two types of green fluorescent protein constructs that either translocate or undergo fluorescence-resonance-energy transfer when local signaling occurs. Broad strategies to measure quantitative, dynamic parameters in signaling networks, together with perturbation approaches, are needed to develop comprehensive models of signaling networks*.

Neuropilins are positive regulators of Hedgehog signal transduction

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.