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Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells

  • Signaling and Cell Physiology
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Abstract

Protein recruitment during endocytosis is well characterized in fibroblasts. Since fibroblasts do not engage in regulated exocytosis, only information about protein recruitment during constitutive endocytosis is provided. Furthermore, the cortical actin of fibroblasts is characterized by stress fibers rather than a thick cortical meshwork. A cell model, which differs in these features, could provide insight into the heterogeneity of protein recruitment to constitutive and exocytosis coupled endocytotic areas. Therefore, this study investigates the sequence of protein recruitment in PC12 cells, a well documented exocytotic cell model with thick actin cortex. Using real time total-internal-reflection fluorescence microscopy it was found that at the plasma membrane steady, but not transient, dynamin-1-EGFP or -mCherry fluorescence spots that rapidly dimmed coincided with markers for constitutive endocytotic such as clathrin-LC-dsRed and transferrin-receptor-pHluorin. Clathrin-LC-dsRed and dynamin-1-EGFP were further used to determine the temporal sequence of protein recruitment to areas of constitutive endocytosis. mCherry- and EGFP-β-actin, Arp-3-EGFP and EGFP-mAbp1 were slowly recruited before the dynamin-1-mCherry fluorescence dimmed, but their fluorescence peaked after the loss of clathrin-LC-dsRed commenced. Furthermore, mCherry-β-actin fluorescence increased before exocytosis, indicating redistribution prior to release. Also, no average dynamin-1-mCherry recruitment was observed within 50 s to regions of exocytosis marked by NPY-mGFP. This indicates that the temporal–spatial coupling between regulated exo-and endocytosis is rather limited in PC12 cells. Furthermore, the time course of the protein recruitment to constitutive endocytotic sites might depend on the subcellular morphology such as the size of the actin cortex.

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Abbreviations

PC12:

pheochromocytoma cell

NPY:

neuropeptide Y

Arp-3:

actin-related protein 3

mAbp1:

mouse actin-binding protein 1

clathrin-LC:

light chain

EGFP:

enhanced green fluorescence protein

mGFP:

monomeric green fluorescence protein

dsRed:

Descosoma coral red fluorescence protein

(c − a):

circle minus annulus

TIRF-M:

total-internal-reflection fluorescence microscopy

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Acknowledgments

I thank Prof. Wolfhard Almers for providing the laboratory space and resources and helpful comments during this study. I thank Mike Hoppa for technical support on mCherry-fusion constructs and many thanks for his comments on the early version of the manuscript. I thank Nick Lesica for critical reading of the manuscript. I thank Prof. Benedikt Grothe for general support. FF was supported by the Max-Planck-Society. This work was supported by NIH grant MH60600.

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  1. Vollum Institute, Oregon Health & Science University, Portland, OR, 97239, USA

    Felix Felmy

  2. Biology II, Department for Neurobiology, Ludwig-Maximilians-University, Großhadernerstr. 2, 82152, Martinsried, Germany

    Felix Felmy

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Correspondence to Felix Felmy.

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Felmy, F. Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells. Pflugers Arch - Eur J Physiol 458, 403–417 (2009). https://doi.org/10.1007/s00424-008-0623-1

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  • DOI: https://doi.org/10.1007/s00424-008-0623-1

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