Volume 7 issue 12 December 2006
Imaging Clathrin Dynamics in D. melanogaster Hemocytes Reveals a Role for Actin in Vesicle Fission
Olexiy Kochubey, Amitabha Majumdar, and Jurgen Klingauf

Supplementary Figures

Supplementary Figure 1. Lateral movement of CCS and mBSA-containing vesicles.
Time-lapse TIRF imaging reveals slow constitutive movement of CCS from the filopodial cell periphery towards the center of footprint (left column, GFP channel). Cargo (mBSA-RhRed) containing vesicles move similarly (middle column, rhodamin red channel) and accumulate cargo within larger compartments centrally inside the cell. Filled arrow heads of same orientation point out the same CCS or cargo vesicles on consecutive images. Overlay images (right column, magnified detail marked by box in left and middle columns) do not show co-localization of CCS and cargo vesicles during their centripetal lateral movement. Empty arrow heads point to center positions of corresponding structures on consecutive images. Scale bars 2 µm. See also Suppl. Movie 1.

Figure 1 (.jpg)

Supplementary Figure 2. Rise of temperature to 34°C does not affect lateral centripetal CCS movement in control hemocytes.
( a ) Maximum projection images obtained from 200 s time lapse series of TIRF images acquired from the same control clc-GFP expressing hemocyte at 25 (left) and 34 °C (right). Unlike in shi^ts2 cells, a temperature increase in control cells does not inhibit centripetal movement of CCS, as the maximum projection images display radial intensity “tracks” resulting from moving CCS. ( b ) Average velocities of moving CCS measured in the same control cells at 25 and 34°C were similar. In total, velocities of n=97 and n=59 CCS in N=6 cells were determined at 25 and 34°C, respectively. Scale bars are 2 µm

Figure 2 (.jpg)

Supplementary Movies

If not separately stated, movies are played at 30x speed and display 2 µm scale bars.

Supplementary Movie 1. Centripetal directed movement of CCS (left, green in overlay) and mBSA-RhRed (middle, red in overlay) containing vesicles.

Movie 1 (.mov)

Supplementary Movie 2. Dynamics of endoplasmic reticulum structures labeled with sqh-EYFP ER marker. The footprint border is outlined in white.

Movie 2 (.mov)

Supplementary Movie 3. Dynamics of early endosomal structures labeled with Rab5-GFP.

Movie 3 (.mov)

Supplementary Movie 4. Dynamics of synaptotagmin-GFP at the footprint.

Movie 4 (.mov)

Supplementary Movie 5. Dynamics of single CCS moving centripetally at hemocyte footprint: persistent CCS, i.e. eventually merging with fluorescence from other structures (left), and disappearing CCS (right). Played at 10x speed, scale bar 1 µm.

Movie 5 (.mov)

Supplementary Movie 6. Effect of actin depolymerization on CCS dynamics (5 µM LatrA)

Movie 6 (.mov)

Supplementary Movie 7. Actin dynamics visualized in hemocytes with actin-GFP and effect upon LatrA application.

Movie 7 (.mov)

Supplementary Movie 8. CCS dynamics in shi^ts2 hemocytes at permissive temperature (25°C, left) and at restrictive temperature (34°C, right).

Movie 8 (.mov)

Supplementary Movie 9. CCS budding from tubular structures in shi^ts2 hemocytes at permissive temperature before heating (left) and during recovery from restrictive temperature (right).

Movie 9 (.mov)