Analyzing Real‐Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules in Endocytosis

Nicholas Hamilton1, Markus C. Kerr1, Kevin Burrage1, Rohan D. Teasdale1

1 ARC Centre in Bioinformatics Institute for Molecular Bioscience The University of Queensland, St. Lucia, Australia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.16
DOI:  10.1002/0471143030.cb0416s35
Online Posting Date:  June, 2007
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Abstract

With the advent of live cell imaging microscopy, new types of mathematical analyses and measurements are possible. Many of the real‐time movies of cellular processes are visually very compelling, but elementary analysis of changes over time of quantities such as surface area and volume often show that there is more to the data than meets the eye. This unit outlines a geometric modeling methodology and applies it to tubulation of vesicles during endocytosis. Using these principles, it has been possible to build better qualitative and quantitative understandings of the systems observed, as well as to make predictions about quantities such as ligand or solute concentration, vesicle pH, and membrane trafficked. The purpose is to outline a methodology for analyzing real‐time movies that has led to a greater appreciation of the changes that are occurring during the time frame of the real‐time video microscopy and how additional quantitative measurements allow for further hypotheses to be generated and tested.

Keywords: endocytosis; video microscopy; endosomal trafficking; membrane trafficking

     
 
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Table of Contents

  • Data and Measurement
  • Conservation of Surface Area
  • Measurement of Volume
  • Flux Across a Membrane, Solute Concentration, and pH Change
  • Pressure, Tension, and Morphology
  • Vesicle Fusion
  • Proportionality and Surface Area
  • Conclusions
  • Acknowledgements
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

  •   FigureFigure 4.16.1 Tubule formation on a vesicle during endocytosis. Flp‐In HEK293 cells stably expressing GFP‐SNX5 were generated as described in Merino‐Trigo et al. (). HEK‐GFP‐SNX5 cell monolayers were serum‐starved overnight before being exposed to 100 ng/ml recombinant EGF ∼1 min before the movie was started. Time‐lapse movies were recorded using an Olympus IX71 inverted microscope equipped with an Olympus 60X oil objective. Images were captured with an IMAGO Super VGA 12 bit 1280 × 1024 pixel CCD camera (T.I.L.L. Photonics). Imaging control and post‐capture image analysis are performed using TILLvisION software and ImageJ v1.31 (see ). The delay between image capture was 10 sec. A single macropinocytic event heavily decorated with GFP‐SNX5 shortly after its formation at the plasma membrane is presented. GFP‐SNX5 is evident on tubular extensions of the limiting membrane of the macropinosome, within the cytosol and on smaller perinuclear puncta reminiscent of early endosomes. Image (A) shows the irregularly shaped vesicle and tubules at time 0. Image (B) is 210 sec later. Significant tubulation has occurred: many long and branched tubules are apparent, and the vesicle appears circular. Image (C) is taken at 780 sec. The vesicle has decreased significantly in area and only a few short tubules are apparent. Bars represents 10 µm. Time‐lapse movie available as Video 1 at http://www.currentprotocols.com.
  •   FigureFigure 4.16.2 The rate of decrease of surface area of the vesicle in The Movie (frames 22 to 83), with an exponential best fit curve (dotted). Surface area is given as four times the area of an ellipse fitted to the vesicle membrane surface.
  •   FigureFigure 4.16.3 Change in a vesicles' surface area and volume as a tubule grows. At time 0 the spherical vesicle has radius rV0. At time t, a cylindrical tubule of length L( t) and radius rT has grown, decreasing the radius of the vesicle to rV( t). If surface area is conserved, then the decrease in surface area of the vesicle will equal the increase in surface area of the tubule, i.e., 4π[ r2V0r2V( t)] = 2π rT L( t). The volume contained in the vesicle is initially 4/3π r3V0. The total volume contained with the vesicle and tubule at time t is 4/3π r3V( t) + π r2T L( t).
  •   FigureFigure 4.16.4 Flux across the membrane. Initially the spherical vesicle has radius r + δ r. At a time δ t later, the radius has decreased by δ r. The volume “between” the vesicle is then 4π r2δ r, and the volume crossing the membrane per unit time is 4π r2δ rt, which becomes −4π r2d r/d t as δ t→0. Hence the volume flux, the volume flow per unit surface area of the vesicle, is −dr/dt.
  •   FigureFigure 4.16.5 Fusion of vesicles. Two spherical vesicles of radius r1 and r2 merge to form a single vesicle of radius r3. If surface area is conserved then the relation 4/3π r12+ 4/3π r22 ≠ 4/3π r32 holds. However, if surface area is conserved volume can not be, i.e., 4/3π r13+ 4/3π r23 ≠ 4/3π r33.
  •   FigureFigure 4.16.6 Vesicle membrane subdomains or patches. The vesicle membrane contains subdomains (shown in grey) on which tubulation is initiated. As the tubule grows the subdomain membrane is drawn into the tubule and the subdomain decreases in size.

Videos

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Internet Resources
   http://rsb.info.nih.gov/ij
  The U.S. National Institutes of Health Web site for ImageJ written by W.S. Rasband and updated from 1997 to 2007.
   http://www.till‐photonics.com/Products/software.php
  The T.I.L.L. Photonics Web site for the TILLvisION image analysis software.
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