Hideji Murakoshi

Phospholipids undergo hop diffusion in compartmentalized cell membrane

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5–100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 μs. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (φ) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-φ compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 μm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

Postsynaptic signaling during plasticity of dendritic spines

Dendritic spines, small bulbous postsynaptic compartments emanating from neuronal dendrites, have been thought to serve as basic units of memory storage. Despite their small size (~0.1 femtoliter), thousands of species of proteins exist in the spine, including receptors, channels, scaffolding proteins and signaling enzymes. Biochemical signaling mediated by these molecules leads to morphological and functional plasticity of dendritic spines, and ultimately learning and memory in the brain. Here, we review new insights into the mechanisms underlying spine plasticity brought about by recent advances in imaging techniques to monitor molecular events in single dendritic spines. The activity of each protein displays a specific spatiotemporal pattern, coordinating downstream events at different microdomains to change the function and morphology of dendritic spines.

Highly sensitive and quantitative FRET–FLIM imaging in single dendritic spines using improved non-radiative YFP

Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their “darkness,” and their ability to act as a FRET acceptor for mEGFP in HeLa cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP–REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by ∼50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFP–mRFP or mEGFP–original REACh pair by ∼50%. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP–sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein–protein interactions in individual dendritic spines in brain slices with high sensitivity.

Microglia contact induces synapse formation in developing somatosensory cortex

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

Ultraspeed single-molecule tracking with <25-μs resolution and electron tomography show that transmembrane proteins and phospholipids in the plasma membrane hop among submicrometer compartments of the same size, probably delimited by the anchored-transmembrane-protein pickets lining the actin-based membrane-skeleton fence, once every 1–58 ms.

Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity

The Rho GTPase proteins Rac1, RhoA and Cdc42 have a central role in regulating the actin cytoskeleton in dendritic spines, thereby exerting control over the structural and functional plasticity of spines and, ultimately, learning and memory. Although previous work has shown that precise spatiotemporal coordination of these GTPases is crucial for some forms of cell morphogenesis, the nature of such coordination during structural spine plasticity is unclear. Here we describe a three-molecule model of structural long-term potentiation (sLTP) of murine dendritic spines, implicating the localized, coincident activation of Rac1, RhoA and Cdc42 as a causal signal of sLTP. This model posits that complete tripartite signal overlap in spines confers sLTP, but that partial overlap primes spines for structural plasticity. By monitoring the spatiotemporal activation patterns of these GTPases during sLTP, we find that such spatiotemporal signal complementation simultaneously explains three integral features of plasticity: the facilitation of plasticity by brain-derived neurotrophic factor (BDNF), the postsynaptic source of which activates Cdc42 and Rac1, but not RhoA; heterosynaptic facilitation of sLTP, which is conveyed by diffusive Rac1 and RhoA activity; and input specificity, which is afforded by spine-restricted Cdc42 activity. Thus, we present a form of biochemical computation in dendrites involving the controlled complementation of three molecules that simultaneously ensures signal specificity and primes the system for plasticity.

The mechanisms underlying the spatial spreading of signaling activity

During the induction of plasticity of dendritic spines, many intracellular signaling pathways are spatially and temporally regulated to co-ordinate downstream cellular processes in different dendritic micron-domains. Recent advent of imaging technology based on fluorescence resonance energy transfer (FRET) has allowed the direct monitoring of the spatiotemporal regulation of signaling activity in spines and dendrites during synaptic plasticity. In particular, the activity of three small GTPase proteins HRas, Cdc42, and RhoA, which share similar structure and mobility on the plasma membrane, displayed different spatial spreading patterns: Cdc42 is compartmentalized in the stimulated spines while RhoA and HRas spread into dendrites over 5-10 μm. These measurements thus provide the basis for understanding the mechanisms underlying the spatiotemporal regulation of signaling activity. Further, using spatiotemporally controlled spine stimulations, some of the roles of signal spreading have been revealed.

Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

Calcium ions function as a booster of chromosome condensation

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca2+, to chromosome condensation in vitro and in vivo. Ca2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca2+. Chromosomes had compact globular structures when exposed to Ca2+ and expanded fibrous structures without Ca2+. Therefore, we have clearly demonstrated a role for Ca2+ in the compaction of chromatin fibres.

A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.