scholarly journals Optimized iLID membrane anchors for local optogenetic protein recruitment

2020 ◽  
Author(s):  
Dean E. Natwick ◽  
Sean R. Collins
Keyword(s):  
Plasma Membrane ◽  
Fusion Proteins ◽  
Protein Localization ◽  
Spatial Scales ◽  
Spatial Dynamics ◽  
Biochemical Networks ◽  
Signaling Networks ◽  
Protein Diffusion ◽  
Spatiotemporal Resolution ◽  
Protein Recruitment

AbstractOptogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long timescales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, and efficient recruitment over wider gene expression ranges. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.SignificanceOptogenetic light-inducible dimer systems, such as iLID, offer the ability to examine cellular signaling networks on second timescales and micrometer spatial scales. Confined light stimulation can recruit proteins to subcellular regions of the plasma membrane, and local signaling effects can be observed. Here, we report alternative iLID fusion proteins that display stronger and more spatially confined membrane recruitment. We also define optogenetic component expression regimes for optimal recruitment and show that slow-diffusing iLID proteins allow more robust recruitment in cell populations with heterogenous expression. These tools should improve the spatiotemporal control and reproducibility of optogenetic protein recruitment to the plasma membrane.

scholarly journals High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits

2020 ◽  
Author(s):  
Jie Wang ◽  
Evelien Mylle ◽  
Alexander Johnson ◽  
Nienke Besbrugge ◽  
Geert De Jaeger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temporal Resolution ◽  
Live Cell Imaging ◽  
Adaptor Protein ◽  
High Temporal Resolution ◽  
Spatiotemporal Resolution ◽  
Membrane Recruitment ◽  
Localization Analysis ◽  
Dynamic Assembly ◽  
Protein Recruitment

AbstractThe TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows down endocytosis and thereby enhances the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, TPC is not recruited to the PM sequentially but as an octameric complex.One sentence summaryLowering the temperature increases spatiotemporal resolution of protein recruitment at the plasma membrane.

scholarly journals Improved synthetic lipidation-based protein translocation system for SNAP-tag fusion proteins

2020 ◽  
Author(s):  
Tatsuyuki Yoshii ◽  
Kai Tahara ◽  
Sachio Suzuki ◽  
Yuka Hatano ◽  
Keiko Kuwata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Proteins ◽  
First Generation ◽  
Protein Translocation ◽  
Protein Localization ◽  
Generation System ◽  
General Applicability ◽  
Protein Targets ◽  
And Function ◽  
In Cells

ABSTRACTThe ability to artificially attach lipids to specific intracellular protein targets would be a valuable approach for controlling protein localization and function in cells. We recently devised a chemogenetic method in which a SNAP-tag fusion protein can be translocated from the cytoplasm to the plasma membrane by post-translationally and covalently conjugating a synthetic lipopeptide in cells. However, the first-generation system lacked general applicability. Herein, we present an improved synthetic lipidation system that enables efficient plasma membrane translocation of SNAP-tag fusion proteins in cells. This second-generation system is now applicable to the control of various cell-signaling molecules, offering a new and useful research tool in chemical biology and synthetic biology.

scholarly journals Mapping High Spatiotemporal-Resolution Soil Moisture by Upscaling Sparse Ground-Based Observations Using a Bayesian Linear Regression Method for Comparison with Microwave Remotely Sensed Soil Moisture Products

2021 ◽  
Vol 13 (2) ◽  
pp. 228
Author(s):  
Jian Kang ◽  
Rui Jin ◽  
Xin Li ◽  
Yang Zhang
Keyword(s):  
Soil Moisture ◽  
Large Scale ◽  
Spatial Scales ◽  
Microwave Remote Sensing ◽  
In Situ Measurements ◽  
Spatial Density ◽  
Linear Regression Method ◽  
Spatiotemporal Resolution ◽  
Pixel Scale

In recent decades, microwave remote sensing (RS) has been used to measure soil moisture (SM). Long-term and large-scale RS SM datasets derived from various microwave sensors have been used in environmental fields. Understanding the accuracies of RS SM products is essential for their proper applications. However, due to the mismatched spatial scale between the ground-based and RS observations, the truth at the pixel scale may not be accurately represented by ground-based observations, especially when the spatial density of in situ measurements is low. Because ground-based observations are often sparsely distributed, temporal upscaling was adopted to transform a few in situ measurements into SM values at a pixel scale of 1 km by introducing the temperature vegetation dryness index (TVDI) related to SM. The upscaled SM showed high consistency with in situ SM observations and could accurately capture rainfall events. The upscaled SM was considered as the reference data to evaluate RS SM products at different spatial scales. In regard to the validation results, in addition to the correlation coefficient (R) of the Soil Moisture Active Passive (SMAP) SM being slightly lower than that of the Climate Change Initiative (CCI) SM, SMAP had the best performance in terms of the root-mean-square error (RMSE), unbiased RMSE and bias, followed by the CCI. The Soil Moisture and Ocean Salinity (SMOS) products were in worse agreement with the upscaled SM and were inferior to the R value of the X-band SM of the Advanced Microwave Scanning Radiometer 2 (AMSR2). In conclusion, in the study area, the SMAP and CCI SM are more reliable, although both products were underestimated by 0.060 cm3 cm−3 and 0.077 cm3 cm−3, respectively. If the biases are corrected, then the improved SMAP with an RMSE of 0.043 cm3 cm−3 and the CCI with an RMSE of 0.039 cm3 cm−3 will hopefully reach the application requirement for an accuracy with an RMSE less than 0.040 cm3 cm−3.

scholarly journals Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giulia Tedeschi ◽  
Lorenzo Scipioni ◽  
Maria Papanikolaou ◽  
Geoffrey W. Abbott ◽  
Michelle A. Digman
Keyword(s):  
Plasma Membrane ◽  
Protein Diffusion ◽  
Ion Diffusion ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Biophysical Characterization ◽  
Kv Channels ◽  
Subunit Stoichiometry ◽  
Spatio Temporal ◽  
Voltage Sensing Domain

AbstractVoltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

scholarly journals Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

2005 ◽  
Vol 169 (6) ◽  
pp. 897-908 ◽  
Author(s):  
Cosima Luedeke ◽  
Stéphanie Buvelot Frei ◽  
Ivo Sbalzarini ◽  
Heinz Schwarz ◽  
Anne Spang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Protein Diffusion ◽  
Ultrastructural Studies ◽  
Bud Neck ◽  
Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

scholarly journals TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

1994 ◽  
Vol 5 (10) ◽  
pp. 1093-1103 ◽  
Author(s):  
A K Rajasekaran ◽  
J S Humphrey ◽  
M Wagner ◽  
G Miesenböck ◽  
A Le Bivic ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Protein Localization ◽  
Evolutionary Relationship ◽  
Cytoplasmic Domain ◽  
Chimeric Proteins ◽  
Madin Darby Canine Kidney ◽  
Surface Domains ◽  
Darby Canine Kidney ◽  
Canine Kidney

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Mechanistic evidence for tracking the seasonality of photosynthesis with solar-induced fluorescence

2019 ◽  
pp. 201900278 ◽  
Author(s):  
Troy S. Magney ◽  
David R. Bowling ◽  
Barry A. Logan ◽  
Katja Grossmann ◽  
Jochen Stutz ◽  
...  
Keyword(s):  
Remote Sensing ◽  
Large Scale ◽  
Spatial Scales ◽  
Gross Primary Production ◽  
Canopy Structure ◽  
Operating Efficiency ◽  
Conifer Forest ◽  
Spatiotemporal Resolution ◽  
Evergreen Forests ◽  
Spectrometer System

Northern hemisphere evergreen forests assimilate a significant fraction of global atmospheric CO2 but monitoring large-scale changes in gross primary production (GPP) in these systems is challenging. Recent advances in remote sensing allow the detection of solar-induced chlorophyll fluorescence (SIF) emission from vegetation, which has been empirically linked to GPP at large spatial scales. This is particularly important in evergreen forests, where traditional remote-sensing techniques and terrestrial biosphere models fail to reproduce the seasonality of GPP. Here, we examined the mechanistic relationship between SIF retrieved from a canopy spectrometer system and GPP at a winter-dormant conifer forest, which has little seasonal variation in canopy structure, needle chlorophyll content, and absorbed light. Both SIF and GPP track each other in a consistent, dynamic fashion in response to environmental conditions. SIF and GPP are well correlated (R2 = 0.62–0.92) with an invariant slope over hourly to weekly timescales. Large seasonal variations in SIF yield capture changes in photoprotective pigments and photosystem II operating efficiency associated with winter acclimation, highlighting its unique ability to precisely track the seasonality of photosynthesis. Our results underscore the potential of new satellite-based SIF products (TROPOMI, OCO-2) as proxies for the timing and magnitude of GPP in evergreen forests at an unprecedented spatiotemporal resolution.

scholarly journals Morphological Alterations of CAD Cells Overexpressing AKT1

2020 ◽  
Vol 26 (S2) ◽  
pp. 1354-1358
Author(s):  
James Wachira
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Survival ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Protein Localization ◽  
Signal Transduction Pathways ◽  
Morphological Alterations ◽  
Cellular Models ◽  
Cad Cells

AbstractCAD cells are neuronal cells used in studies of cell differentiation and in cellular models of neuropathology. When cultured in differentiation medium, CAD cells exhibit characteristics of mature neurons including the generation of action potential. In addition to being a central signaling kinase in cell survival, AKT1 plays important roles in the nervous system including neuroplasticity and this study examined the localization of exogenous AKT1 in CAD cells. Neuropeptides modulate many signal transduction pathways and melacortins are implicated in regulating growth factor signal transduction pathways, including the PI3K/AKT pathway. AKT1-DsReD was transfected into CAD cells that were stably expressing melanocortin 3-receptor-GFP (MC3R-GFP), a G-protein coupled receptor. The cells were imaged with confocal microscopy to determine the fluorescent protein localization patterns. AKT1-DsRed was predominantly localized in the cytoplasm and the nucleus. Further, expression of exogenous AKT1 in these cell lines led to morphological changes reminiscent of apoptosis. As expected, MC3R-GFP localized to the plasma membrane but it internalized upon cell stimulation with the cognate ligand. In limited areas of the plasma membrane, AKT1-DsRed and MC3R-GFP were colocalized. In conclusion, quantitative studies to understand the role of relative levels of AKT1 in determining cell survival are needed.

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