MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING

ABSTRACTSingle-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in “hotspots” on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of individual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.

Download Full-text

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

Scientific Reports ◽

10.1038/s41598-021-90002-2 ◽

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.

Download Full-text

Single-Molecule Characterization of the Dynamics of Calmodulin Bound to Oxidatively Modified Plasma-Membrane Ca2+-ATPase†

Biochemistry ◽

10.1021/bi050488m ◽

2005 ◽

Vol 44 (33) ◽

pp. 11074-11081 ◽

Cited By ~ 15

Author(s):

Kenneth D. Osborn ◽

Asma Zaidi ◽

Ramona J. Bieber Urbauer ◽

Mary L. Michaelis ◽

Carey K. Johnson

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Oxidatively Modified

Download Full-text

Fluorescence Fluctuation Spectroscopy Enables Quantification of Potassium Channel Subunit Dynamics and Stoichiometry

10.21203/rs.3.rs-121439/v1 ◽

2020 ◽

Author(s):

Giulia Tedeschi ◽

Lorenzo Scipioni ◽

Maria Papanikolaou ◽

Geoffrey Abbott ◽

Michelle Digman

Keyword(s):

Plasma Membrane ◽

Protein Diffusion ◽

Ion Diffusion ◽

Fluorescence Fluctuation Spectroscopy ◽

Biophysical Characterization ◽

Kv Channels ◽

Subunit Stoichiometry ◽

Spatio Temporal ◽

Voltage Sensing Domain

Abstract Voltage-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.

Download Full-text

Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome

10.1101/267930 ◽

2018 ◽

Author(s):

Charlotte Floderer ◽

Jean-Baptiste Masson ◽

Elise Boiley ◽

Sonia Georgeault ◽

Peggy Merida ◽

...

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Viral Genome ◽

Virus Assembly ◽

Host Cells ◽

Spherical Particles ◽

Gag Protein ◽

Numerous Cell ◽

Spatio Temporal ◽

Hiv 1

Monitoring virus assembly dynamic at the nanoscale level in host cells remains a major challenge. Human Immunodeficiency Virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.

Download Full-text