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

Calcium ions function as a booster of chromosome condensation

Phengchat, R., Takata, H., Morii, K. et al. Calcium ions function as a booster of chromosome condensation. Sci Rep 6, 38281 (2016). https://doi.org/10.1038/srep38281

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

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion

LOV2-based photoactivatable CaMKII and its application to single synapses: Local Optogenetics

Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the “high spatial resolution” of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as “Local Optogenetics”, which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII

Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors

Fluorescence imaging for monitoring the colocalization of two single molecules in living cells

ABSTRACT The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/ background ratios, we found that two single molecules in dual color can be colocalized to within 64–100 nm (68–90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules

Schematic diagram of CaMKIIα FRET sensor evolution.

<p><b>(A)</b> The CaMKIIα fluorescence resonance energy transfer (FRET) sensor (Camuiα) consists of FRET donor (mEGFP) and acceptor fluorophores (sREACh) and full-length CaMKIIα, which contains kinase, regulatory, and association domains. For improving the folding efficiency of the sensor, the association domain was amplified by error-prone PCR and subcloned into a bacterial expression vector. Subsequently, the ligated DNA was transformed in <i>Escherichia coli</i> to make a library. Bright red colonies were isolated from the library and analyzed by DNA sequencing. The identified mutations were then incorporated into the FRET sensor, leading to an increase in its folding efficiency. This scheme is based on an assumption that the folding efficiency of the association domain is coupled to that of mCherry. <b>(B)</b> The crystal structure of the association domain of human CaMKIIβ7 (PDB code: 3SOA). Note that this sequence is identical to the corresponding region in rat CaMKIIα (345–474) used in this study. The positions of four mutations (F394L, I419V, A430T, and I434T) found in this study are shown in red.</p