hahn lab

cell cinema

Engineered allosteric control of protein activity - Loopology and RapR approaches for optogenetic and chemogenetic control

3 cells showing activation by Rapr analogs of the Src family: Fyn, Src and Lyn.

RapR analogs of Src family kinases enable specific activation of closely related family members (Fyn, Src, and Lyn). Each produces a distinct phenotype.

These 3 videos show:

Top - Fyn (spreading)
Middle - Src (spreading followed by movement)
Bottom - Lyn (suprising kinked extensions)

Video by Pei Hsuan Chu
PNAS, 111(34):12420-25, 2014

cell showing activation of RapR-Src generates cells preading and adheasion dynamics

Activation of RapR-Src generates cell spreading, accompanied by adhesion dynamics

Video by Andrei Karginov
Nature Chem Biol, 10(4):286-90, 2014

a cell

Loopology: Upon photo-inhibition, Src translates to focal adhesions and induces polarized movement.

Video by Onur Dagliyan
Science, 354(6318):1441-1444, 2016

2 cells

Loopology: Photo-inhibition of Vav2 produces reversible contraction

Video by Onur Dagliyan
Science, 354(6318):1441-1444, 2016

cell

Loopology: Combined photo-activation (PA-Vav2) and photo-inhibition (PI-Rac1) in the same cell

Video by Onur Dagliyan
Science, 354(6318):1441-1444, 2016

3 images of a cell showing Rac1 photo inhibition

Loopology: Rac1 photo-inhibition. Irradiation induces rapid ruffling followed by cessation of ruffling.

Video by Onur Dagliyan
Science, 13:755-758, 2016

Loopology: Vav2 photo-inhibition. Reversible inhibition of Vav2 produces blebbing.

Video by Onur Dagliyan
Science, 13:755-758, 2016

PA-Rac1

moving laser causes cell protrusions

PA-Rac: Moving laser spot induces Rac1 activation, generating localized protrusion

Video by Yi Wu
Nature, 46:104-110, 2009

PA-Rac: Pulses of light on one spot along the cell edge (red circle) activate Rac1 locally, producing first ruffles and then a protrusion

Video by Yi Wu
Nature, 461:104-110, 2009

PA-Rac: HEK293 cell follows a light spot that activates Rac1

Video by Yi Wu
Nature, 461:104-110, 2009

Zebrafish spelling Rac - wiggly shaped letters R, A and C.

PA-Rac: Fluorescent neutrophils in living zebrafish spelling "Rac"

Video by our collaborators, Sa Kan Yoo and Qing Deng, in the Huttenlocher Lab
Dev. Cell, 18:226-23, 2010

blue cell with with green a nucleus surrounded by red theny yellow then green then blue bands of color against a black background.

NIGMS Biomedical Beats: "Cool video: Leading Cells with light"

Video captions and narration by NIH, images by Yi Wu
NIGMS Biomedical Beats, Sept, 2009

Lovetrap and miscellaneous optogenetics

LOVTRAP: reversible sequestration of GFP at mitochondria

Video by Hui Wang
Nature Methods, 13:755-758, 2016

LOVTRAP: activation of VaV2 produces cell oscillations

Video by Hui Wang
Nature Methods, 13:755-758, 2016

Optogenetic control of nuclear import. Control of NLS/NES exposure

Video by our collaborators in the Kuhlman Lab
Nature Chem. Biol., 12(6): 399-401, 2016.

Genetically encoded biosensors

RhoA activation versus localization in a moving fibroblast

Video by Olivier Pertz and Louis Hodgson
Nature, 440:1069-1072, 2006

Rac1 activation in a motile fibroblast

Video by Christopher Welch
Nature, 461:104-110, 2009

Activation of Rap1 at tips of dorsal ruffles. 3D analysis using ImageTank

Video by Ellen O'Shaughnessy
J. Cell Biol., 218(9):3153-3160, 2019

Rap1 activity bridges ventral ECM interactions and dorsel ruffles. 3D visualization using ImageTank software

Video by Ellen O'Shaughnessy
J. Cell Biol., 218(9):3153-3160, 2019

3D view of Rap1 activation in moving fibroblast using ImageTank

Video by Ellen O'Shaughnessy
J. Cell Biol., 218(9):3153-3160, 2019

blue cell with a yellow and green nucelus with many purple.

GEF-H1 is held in the off state on microtubules and released at microtubule tips for localized activation. Microtubules (L), GEF-H1 activity (R)

Video by Mihai Azoitei
J. Cell Biol., 218 (9):3077–3097, 2019

blue cell with a yellow and green nucelus with many purple.

Multiplexed imaging of GTPases and GEFs

Video by Daniel J. Marston
Nature Chem. Biol., 16(8):826-833,2020.

SPECTR (Small Peptide Exposure for Conformation Tracking)

Images of individual molecues colored red, green and yellow in a living cell. The yellow is the Src molecule which shows a yellow biosensor when in its open conformation.

Conformational changes of individual molecules within living cells. Src molecules turn yellow when in open conformation.

Video by Bei Liu
Cell, 2021:Oct11; doi.org/10.1016/j.cell.2021.09.026
Initially named binder/tag

A blue cell with dots of green, dark blue and yellow, mostly along the cell edge, which are proteins with biosensors attached, that change color as the proteins within the cell, do different activities which cause its conformation to change.

Making a FRET biosensor using SPECTR. Src activation during cell motility.

Video by Bei Liu
Cell, 2021:Oct11; doi.org/10.1016/j.cell.2021.09.026

Biosensors based on environment-sensing dyes

Cdc42 activation in RhoA-inhibited cell; meroCBD biosensor

Video by Perihan Nalbant and Louis Hodgson
Science, 305:1615-1619, 2004

protein structure of phospho-ERK2 with merocyanine dye attached

Merocyanine dye on phospho-ERK2

Video by Alexei Toutchkine and Brenda Temple

blue cell with many colored biosensors around the edge of the cell indicating Src activation in macropinocytic vesicles.

Src activation in macropinocytic vesicles.

Video by Akash Gulyani
Nature Chem Biol, 7(7):437-44, 2011

Podosome biology

Macrophage forms a ring of podosomes (purple dots) when contacting a micro patterned circle of substrate antibodies (green).

Video by Takashi Watanbe
Nature Communications, 13:4363, 2022

Microtubule trapped within a ring of podosomes (at approximately 5 o’clock)

Video by Shiqiong Hu
Nature Communications, 13:4363, 2022


Podosome Structures

Cartoon showing overall structure with multiple proteins.

Podsome structure and dynamics


3D-SIM projection of F-actin and myosin II in a ring of podosomes

Video by Shiqiong Hu
Nature Communications, 13:4363, 2022.

 

 

Cartoon showing protein positions

Proteins (top to bottom):
IgG, FcγRIIA, F-actin, Paxillin, Talin, Myosin II

Cartoon by Megan Kern
Nature Communications, 13:4363, 2022.

Image analysis and modeling

Sampling windows track the cell edge - localized assessment of biosensor correlation with cell behavior

Video by our collaborators in the Danuser lab
Nature, 461:99-103, 2009

orange cell with automated line tracking graph next to it

Automated tracking of cell morphology. Simple 2D trace captures morphodynamics over time

Video by our collaborators in the Elston and Tsygankov labs
PNAS, 111(34):12420–12425, 2014

CellGeo package accurately measures dimensions and morphodynamics of complex cell boundaries

Video by our collaborators in the Elston and Tsygankov labs
J. Cell Biol., 204(3):443-460, 2014

Danuser lab analysis of coordination between GEF-H1 activity and cell protrusion

Video by Mihai Azoitei
J. Cell Biol., 218(9):3077–3097, 2019

Miscellaneous

Movie 1 3 cells showing activation by Rapr analogs of the Src family: Fyn, Src and Lyn. Movie 2 3 cells showing activation by Rapr analogs of the Src family: Fyn, Src and Lyn.
 

Megakaryocyte proplatelet extensions

Videos by Ellen O'Shaughnessy and Ana Nogueria
Lab Chip, 22(1): 136–147, 2021.

Cytotoxic T lymphocyte contacts virally infected fibroblast. This leads to synapse formation, accumulation of cytotoxic granules at the interface, injection of granule contents into the fibroblast, and finally fibroblast apoptosis. (old movie captured using video tape – the good old days)

Video by Klaus Hahn, Robin DeBiasio, Greg LaRocca, D. Lansing Taylor, and Michael Oldstone
Virology, 201(2):330-340, 1994

2 green colored cells have bright round areas moving around inside them

GFP Vinculin reveals adhesions and dorsal ruffling

Video by Louis Hodgson

Retrograde Transport

Video by Noah Sciaky

Migrating fibroblasts

Video by Feimo Shen


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