TY - CHAP
T1 - Quantitative image analysis of the spatial organization and mobility of caveolin aggregates at the plasma membrane
AU - Hirama, Takashi
AU - Das, Raibatak
N1 - Funding Information:
We thank Gregory D. Fairn, Keenan Research Centre for Biomedi-cal Science, St. Michael’s Hospital, Toronto, Canada, and Sergio Grinstein, Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada, for their support and for guiding the research presented here. This work is supported in part by Kurozumi Medical Foundation and Tokyo-Hokenkai Byotai-Seiri Laboratory.
Funding Information:
We thank Gregory D. Fairn, Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Canada, and Sergio Grinstein, Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada, for their support and for guiding the research presented here. This work is supported in part by Kurozumi Medical Foundation and Tokyo-Hokenkai Byotai-Seiri Laboratory.
Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2020.
PY - 2020
Y1 - 2020
N2 - Caveolins are integral membrane proteins that are the principal structural component of caveolae. Newly synthesized caveolin self-associates into oligomers that further assemble into higher-order structures. Imaging fluorescently labeled caveolin at the plasma membrane with total internal reflection fluorescence (TIRF) microscopy reveals a spatially heterogeneous distribution with aggregates of various sizes. In this chapter, we present a set of image-processing tools to quantify the spatial organization and mobility of caveolin aggregates seen in TIRF images. We apply a spot detection algorithm to identify punctate features on multiple length scales, and computationally estimate the area and integrated fluorescence signal of each detected feature. We then partition the original image into two disjoint sets: one containing pixels within punctae, and the other containing pixels on the rest of the plasma membrane. From these partitions, we estimate the relative fraction of caveolin that is punctate versus diffuse. Finally, we analyze the mobility of caveolin aggregates by tracking them and classify individual trajectories as diffusive or subdiffusive using a moment scaling spectrum analysis. Together, these analyses capture multiple facets of caveolin organization and dynamics. To demonstrate their utility, we quantify the distribution of fluorescent Caveolin 1 stably transfected in HeLa cells. We analyze cells at baseline and after being exposed to the anesthetic Dibucaine that is known to scramble membrane phospholipids. Our analysis shows how this perturbation dramatically alters caveolin aggregation and mobility.
AB - Caveolins are integral membrane proteins that are the principal structural component of caveolae. Newly synthesized caveolin self-associates into oligomers that further assemble into higher-order structures. Imaging fluorescently labeled caveolin at the plasma membrane with total internal reflection fluorescence (TIRF) microscopy reveals a spatially heterogeneous distribution with aggregates of various sizes. In this chapter, we present a set of image-processing tools to quantify the spatial organization and mobility of caveolin aggregates seen in TIRF images. We apply a spot detection algorithm to identify punctate features on multiple length scales, and computationally estimate the area and integrated fluorescence signal of each detected feature. We then partition the original image into two disjoint sets: one containing pixels within punctae, and the other containing pixels on the rest of the plasma membrane. From these partitions, we estimate the relative fraction of caveolin that is punctate versus diffuse. Finally, we analyze the mobility of caveolin aggregates by tracking them and classify individual trajectories as diffusive or subdiffusive using a moment scaling spectrum analysis. Together, these analyses capture multiple facets of caveolin organization and dynamics. To demonstrate their utility, we quantify the distribution of fluorescent Caveolin 1 stably transfected in HeLa cells. We analyze cells at baseline and after being exposed to the anesthetic Dibucaine that is known to scramble membrane phospholipids. Our analysis shows how this perturbation dramatically alters caveolin aggregation and mobility.
KW - Caveolin
KW - Moment scaling spectrum
KW - Spatial heterogeneity
KW - Spot detection
KW - TIRF
UR - http://www.scopus.com/inward/record.url?scp=85086685577&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85086685577&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-0732-9_5
DO - 10.1007/978-1-0716-0732-9_5
M3 - Chapter
C2 - 32548818
AN - SCOPUS:85086685577
T3 - Methods in Molecular Biology
SP - 53
EP - 62
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -