Clathrin-mediated endocytosis (CME) is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. Currently, there are two models describing membrane bending during the formation of clathrin cages: the first involves the deposition of all clathrin molecules to the plasma membrane, forming a flat lattice prior to membrane bending, whereas in the second model, membrane bending happens simultaneously as the clathrin arrives to the site to form a clathrin-coated cage. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorophores (actin filaments labeled with green phalloidin and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. An extensive statistical survey of many hundreds of CME events, at various stages of progression, are observed via this method, allowing inferences about the dominant mechanisms active in CME in SKMEL cells. Results indicate a mixed model incorporating aspects of both the aforementioned mechanisms for CME.
Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems
in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single
biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in
quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by
selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is
difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response
time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for
two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that
employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them.
Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This
technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective
excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins
mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of twophoton
FRET. This work paves the way for two-photon FRET stoichiometry.
A complete understanding of cellular behavior will require precise temporal and spatial measurement of protein-protein interactions inside living cells. FRET Stoichiometry (Hoppe, A.D. et al., 2002 Biophys. J. 83:3652) has been used to measure the timing and spatial organization of protein-protein interactions in cells expressing yellow fluorescent protein (YFP)-labeled proteins and cyan fluorescent protein (CFP)-labeled proteins. However, all FRET data
collected in a single plane of a widefield microscope is a distorted 2D representation of a 3D object. Here we show that image blurring in the widefield microscope dramatically reduces sensitivity and spatial discrimination of FRET-based measurements of protein interactions. We present an algorithm for 3D restoration and calculation of FRET data that greatly increases signal-to-noise ratio and accuracy. The approach uses maximum likelihood deconvolution to quantitatively reassign out-of-focus light in 3D-FRET data sets. FRET Stoichiometry calculations performed on test constructs of linked YFP-CFP produced images that displayed uniform apparent FRET efficiencies (both EA and ED) and molar ratio of 1. 3D images of cells expressing free YFP and free CFP indicated apparent FRET efficiencies of 0%. Furthermore, 3D-FRET Stoichiometry imaging of the interaction of activated YFP-Rac1 with CFP-PBD in living cells produced superior detail with maximal apparent FRET efficiencies that were consistent with in vitro data. Together, these data demonstrated 3D-FRET Stoichiometry could accurately measure the fractions of interacting molecules and their molar ratios with high 3D spatial resolution.