Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development.
Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin’s head and tail. FRET efficiency decreases with increased tension across vinculin.
Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones.
Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod (28.5 ± 3.6 % ) in growth cones was higher than the mean FRET efficiency of VinTS (24.6 ± 2 % ) and VinTL (25.8 ± 1.8 % ) (p < 10 − 6). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant (p < 10 − 3), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod.
Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.
Vinculin is a known key regulator of focal adhesions; it undergoes tension in the locations of attachment to the extracellular matrix. In this study, we explore the use of a vinculin tension FRET probe to investigate vinculin tension within neurons. A critical component of neuronal growth is migration, which is dependent on the mechanical cues between the cells and the extracellular matrix. An understanding of tension variation within the neuron may help us understand mechanisms of neurogenesis. To study these forces, we use a previously developed molecular tension sensor, which consists of an elastic linker, TSMod, a 40-amino-acid-long peptide inserted between teal fluorescent protein (mTFP1) and mVenus. The vinculin tension sensor, VinTS, consists of TSMod embedded between the Vinculin head and tail. When under tension, VinTS will exhibit a lower fluorescence resonance energy transfer (FRET) efficiency between mTFP1 and mVenus. Cortical neurons were isolated from embryonic rat brains and cultured on glass coverslips coated with poly-D-lysine and laminin. The neurons were transfected with TSMod (the unloaded tension sensor) or VinTS. Neurons expressing TSMod are used as the experiment’s control group since TSMod on its own is not affected by vinculin tension. The mean FRET efficiency of 171 TSMod and 127 VinTS expressing neurons was 27.08 ± 4.98%, and 22.86 ± 3.98%, respectively. The FRET efficiency of VinTS was significantly lower than that of TSMod (p = 6.6e15 by Welch’s t-test). These results support the feasibility of using the VinTS probe in neurons and provide a first assessment of VinTS FRET efficiency in neurons. The lower FRET efficiency of VinTS compared with TSMod suggests that VinTS may be under tension in neurons. However, additional studies are required to further characterize these results.
An efficient sensitivity analysis approach for quantum nanostructures is proposed. The imaginary time propagation method (ITP) is utilized to solve the Time Dependent Schrödinger’s Equation (TDSE). Using this method, an extraction of all the modes and their sensitivity with respect to all the design parameters have been performed with minimal computational effort. The sensitivity analysis is performed using the Adjoint Variable Method (AVM) and results are comparable to those obtained using Central Finite Difference Method (CFD) applied directly on the response level.
Using plasmonic waveguide for interconnects application is very promising direction to achieve high density integration
a good size compatibility with electronic devices. Thus, proposing compact and efficient functional plasmonic devices is
or prime essential to achieve the required system functionalities. Power splitters are widely used as one of the important
component of the optical interconnects and integrated photonic and plasmonics devices. We propose a simple, ultra
compact and wideband balanced power divider. The advantage of this device is compactness and ability to split the
power over wideband with negligible imbalance. The device is based on plasmonic slot waveguide configuration and has
submicron total foot print. To achieve the proposed optimized design, a simple and novel analytical model is utilized for
modelling the behavior or any plasmonic structure using circuit model.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.