Imaging and Defining Emergent Behaviors of the Immune Response

T cell Motility

Actin and Myosin Dynamics in Cell Motility:

T cells are motile via at least two distinct mechanisms. The first, which we term ‘Sliding’ is very similar to how mesenchymal cells move on glass or coated surfaces and involves a single contact which translates continuously, akin to a tank tread. The second, involves coordinated placements of one or more contacts onto the substrate in sequence, akin to how humans translate weight between alternating legs, transferring force to the floor to move forward (thus ‘Walking’). We believe the ‘Walking’ mode is more likely the physiological reality in vivo, excepting when integrin levels are really high or, when an intracellular motor protein called myosinIIA is deactivated, for example during TCR signaling. Walking is a faster mode than Sliding whereas the latter, with increased surface contact, is likely to give rise to greater and more prolonged surface contact, which we believe to be important to increase sensitivity to ligands in the environment.

‘Walking’ T cell motility: Contact zone dynamics of a 'walking' control cell. 
D10 T cells co-transfected with control-shRNA, and actin-GFP plasmids were plated on casein blocked coverslips and imaged by TIRF microscopy at 2 sec intervals for 5 min. Brightfield images are overlayed with a pseudo-colored TIRF image of the actin-GFP fluorescence. From Jacobelli et al. JI 2009.

‘Sliding’ T cell Motility: This mode is enhanced by either increased levels of integrin ligands OR deactivation of MyosinIIA.

Contact zone dynamics of a 'sliding' control cell on ICAM-1. 
D10 T cells co-transfected with control-shRNA, and actin-GFP plasmids were plated on ICAM-1 coated coverslips and imaged by TIRF microscopy at 2 sec intervals for 5 min. Brightfield images are overlayed with a pseudo-colored TIRF image of the actin-GFP fluorescence. From Jacobelli et al. JI 2009.

Contact zone dynamics of a 'sliding', MyoIIA depleted cell. 
D10 T cells co-transfected with MyoIIA-shRNA, and actin-GFP plasmids were plated on casein blocked coverslips and imaged by TIRF microscopy at 2 sec intervals for 5 min. Brightfield images are overlayed with a pseudo-colored TIRF image of the actin-GFP fluorescence. From Jacobelli et al. JI 2009.

MyosinIIA localization suggests a coordinated population of Walking ‘footprints’ with actin followed by Myosin and a critical role in extinguishing individual ‘footprints’ during walking.

MyoIIA dynamics in the contact zones of a 'walking' cell. 
D10 T cells co-transfected with MyoIIA-GFP and actin-mCherry were plated on casein blocked coverslips and imaged by TIRF microscopy at 2 sec intervals for 5 min. In the left panel brightfield images are overlayed with the TIRF images of the MyoIIA-GFP fluorescence (in green) and actin-mCherry fluorescence (in red). In the right panel only the TIRF images of the MyoIIA-GFP fluorescence (in green) and actin-mCherry fluorescence (in red) are shown. From Jacobelli et al. JI 2009.

MyoIIA cluster coalescence during contact zone contraction in a 'walking' cell. 
Tracking of selected MyoIIA clusters in the contact zone of a 'walking' cell. The overall paths of the clusters are overlayed in red and transition to yellow as each cluster moves along its path. From Jacobelli et al. JI 2009.

The importance of Myosin IIA in modulating the motility mode and increasing motility rate is evident in vivo: Naïve MyoIIA-deficient T cells have reduced intra-lymph node migration. Representative movie of interstitial migration in vivo of control T cells (green) and MyoIIA cKO T cells (red). Naïve CD8+ T cells were purified by negative selection from control and MyoIIA cKO mice, then labeled with either CFSE or CMTMR, mixed at a 1:1 ratio and injected intravenously into recipient mice. Popliteal, axillary and inguinal lymph nodes were isolated 18h after transfer and imaged by time-lapse 2-photon laser scanning microscopy. The duration of the timelapse is 30 min at 20 sec intervals between frames. The tracks were obtained using Imaris software and the latest trailing 20 frames are shown. The grid spacing is at 20 mm. From Jacobelli et al. NI 2010.


The Role of Confinement in Regulating Speed:

3 Dimensional Confinement regulates Motility Rate.
This movie shows cell migration in three channels of differing dimension. Motility is maximal at intermediate channel size. We interpret this as cell motility being frictionally-limited when diameters are small and cells are dragging lots of their surface along their environment and being inefficient due to excess actin polymerization perpendicular to the direction of propagation when excess space is confined. In the ‘Goldilocks’ zone (moderate confinement), cells can modulate their interaction with their environment to optimize directional motion while limiting frictional effects. From Jacobelli et al. Nature Immunology, 2010.

‘Walking’ in Microchannels.
This movie demonstrates migration within microchannels, using TIRF imaging to fluorescently highlight the contacts of cells on the glass. This demonstrates ‘walking’ motility. From Jacobelli et al. Nature Immunology, 2010.

‘Sliding’ in Microchannels, in the presence of Blebbistatin to Inhibit Myosin II.
This movie demonstrates migration within microchannels, using TIRF imaging to fluorescently highlight the contacts of cells on the glass. This demonstrates ‘sliding’ motility in the absence of MyosinII function. From Jacobelli et al. Nature Immunology, 2010.


Septin Regulates T cell Shape and Coordinated Motility:

T cells contain a septin cytoskeleton, composed of arrays of individual septin monomers that aggregate into filaments. These appear to stabilize the cortex against blebbing and modify cell shape. Loss of this cytoskeleton, for example by shRNA-mediated depletion of the critical Septin7 protein, results in multiple abnormal phenotypes and results in discoordinated and inefficient and slow motile rates (Tooley et al. NCB 2008)

Three-dimensional renderings of annular and cortical Septin bundles.
D10 T cells, stained with anti-Septin7 and subjected to confocal imaging were rendered using a maximal intensity projection algorithm to generate a three-dimensional reconstruction. Two cells are shown. From Tooley et al. NCB 2008.

Membrane blebbing in Sept7KD D10 T cells.
Time-lapse images were acquired at 5–10 sec intervals for at least 10 min in cells depleted of Septins. Membrane blebbing is indicated by arrows during the movie. Imaging was performed in 0.25% low-melting point agarose. From Tooley et al. NCB 2008.

Excess protrusions in Sept7KD D10 T cells. 
Tine-lapse images were acquired at 5–10 second intervals for at least 10 min in cells depleted of Septins. Excess protrusions were observed for two different cells during the movie and are indicated by arrows. Imaging was performed in 0.25% low-melting point agarose. From Tooley et al. NCB 2008.

 

 

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