Abstract
Despite a large body of publications on endocytosis, only a few studies have focused on its spatial organization. To study how endocytosis is related to distinct cellular sites, we combine cell normalization by the “micropatterning technique” with the quantification of spatial organization by “probabilistic density mapping.” Micropatterns of extracellular matrix proteins impose adhesive and non-adhesive areas to cultured cells and allow the control of adhesion geometry, shape, and cell organization. Probabilistic density maps provide a visual summary for 3D localization of the structures of interest and enable the extraction of robust statistics for quantification of cellular organization. Here, we provide a method to analyze and compare the spatial distribution of endocytosed ligands in micropatterned cells. This approach permits to establish the role of cellular adhesion on uptake mechanisms and to address the potential function of predefined sites of endocytosis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rappoport J, Simon S (2003) Real-time analysis of clathrin-mediated endocytosis during cell migration. J Cell Sci 116:847–855
Cao H, Krueger E, McNiven M (2011) Hepatocytes internalize trophic receptors at large endocytic “Hot Spots”. Hepatology 54:1819–1829
Gaffield M, Tabares L, Betz W (2009) Preferred sites of exocytosis and endocytosis colocalize during high- but not lower-frequency stimulation in mouse motor nerve terminals. J Neurosci 29:15308–15316
Gumbiner B (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345–357
Thery M, Racine V, Piel M et al (2006) Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc Natl Acad Sci U S A 103:19771–19776
Schauer K, Duong T, Bleakley K et al (2010) Probabilistic density maps to study global endomembrane organization. Nat Methods 7:560–566
Grossier JP, Xouri G, Goud B, Schauer K (2014) Cell adhesion defines the topology of endocytosis and signaling. EMBO J 33(1):35–45
Schneider C, Rasband W, Eliceiri K (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
R Development Core Team (2013) R: a language and environment for statistical computing. Vienna, Austria
Duong T (2009) ks: Kernel smoothing, R package version 1.6.3
Azioune A, Storch M, Bornens M, Théry M, Piel M (2009) Simple and rapid process for single cell micro-patterning. Lab Chip 9 1640–1642
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Grossier, JP., Goud, B., Schauer, K. (2014). Probabilistic Density Maps to Study the Spatial Organization of Endocytosis. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0944-5_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0943-8
Online ISBN: 978-1-4939-0944-5
eBook Packages: Springer Protocols