Tag Archive for: Cytoskeleton

Following Tubulin Dynamics during Spindle Formation using Photoconversion of mEos2

To analyze the tubulin dynamics during spindle formation, Drosophila S2 cells were transfected with mEos2-labled tubulin. By local irradiation with UV laser light (405nm) the green fluorescent mEos2 photoconverts to a red fluorescent dye. In contrast to photobleaching, where the fluorescent signal is locally depleted, the photoswitching allowed the independent observation of two different tubulin fractions in space and time.


 

 

 

 

 

Movie1: Photoconversion of mEos2-labeled tubulin in the spindle of a Drosophila S2 cell. The video is sped up 20-fold from real time.

Setup:

  • Microscope: Standard widefield microscope
  • Objective: 100x NA 1.4
  • 405 & 473 nm diode laser

Rapp OptoElectronic Components:

  • UGA-40 – point scanning device (integrated in µ-manager)

 

 

Data taken from:
Kurt’s Microscopy Blog
http://nic.ucsf.edu/blog/2014/04/photobleaching-and-photoactivation/

Kurt Thorn (1) & Nico Stuurman (2)

(1) Nikon Imaging Center (NIC) at University of California – San Francisco (UCSF)
(2) Vale Lab at University of California – San Francisco (UCSF)

Cutting Actin Bundles in Single Cells

Actin bundles are the force generating part of a cell, and thus are major players during cell-mechanical processes like cell migration. By cutting individual actin fibers and quantifying the corresponding retraction, the underlying physical properties, like tension and internal forces can be determined. Additionally, by cutting force-generating stress-fibers during cell migration, cell responses, like loss of polarity and reorientation, can be observed.


 

 

 

 

Movie1: Migrating Keratinocyte. Actin network was stained using life-act (shown in black; inverted LUT). Individual actin bundles were cut to analysis retraction as measure of tension within the actin network.

 

Setup:

  • Zeiss Observer Spinning Disk
  • 40x EC Plan-Neofluar NA 1.3 (oil)

Rapp OptoElectronic components:

  • UGA-42 Caliburn 355/42 (pulsed laser, 355nm, 1KHz, 42µJ/pulse)

Data from:

Demo data acquired together with Prof. Merkel’s group (ICS-7; Research Center Jülich) at Prof. Großhans’ Lab (Developmental Biochemistry; University of Göttingen).