scholarly journals Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

2021 ◽  
Author(s):  
Daan Vorselen ◽  
Sarah R. Barger ◽  
Yifan Wang ◽  
Wei Cai ◽  
Julie A. Theriot ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Traction Force ◽  
Light Sheet ◽  
Force Generation ◽  
Actin Dynamics ◽  
Force Microscopy ◽  
Light Sheet Microscopy ◽  
Macrophage Phagocytosis ◽  
Actin Protrusions

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely mediated by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that are interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, suggesting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings suggest a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

scholarly journals Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Daan Vorselen ◽  
Sarah R Barger ◽  
Yifan Wang ◽  
Wei Cai ◽  
Julie A Theriot ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Traction Force ◽  
Light Sheet ◽  
Force Generation ◽  
Actin Dynamics ◽  
Force Microscopy ◽  
Light Sheet Microscopy ◽  
Macrophage Phagocytosis ◽  
Actin Protrusions

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

scholarly journals Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

2021 ◽  
Author(s):  
Yukako Nishimura ◽  
Shidong Shi ◽  
Qingsen Li ◽  
Alexander D. Bershadsky ◽  
Virgile Viasnoff
Keyword(s):  
Contractile Activity ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Traction Force ◽  
Stress Fiber ◽  
Force Generation ◽  
Stress Fibers ◽  
Force Microscopy ◽  
Fiber Dynamics ◽  
The Relationship

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation.

scholarly journals Spatiotemporal co-dependency between macrophages and exhausted CD8+ T cells in cancer

2021 ◽  
Author(s):  
Kelly Kersten ◽  
Kenneth H Hu ◽  
Alexis J Combes ◽  
Bushra Samad ◽  
Tory Harwin ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Spatially Resolved ◽  
Hypoxic Conditions ◽  
Synaptic Interactions ◽  
Positive Feedback Circuit ◽  
Major Impediment

T cell exhaustion is a major impediment to anti-tumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here we show that the biology of tumor-associated macrophages (TAM) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAM reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique long-lasting antigen-specific synaptic interactions that fail to activate T cells, but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

Multiphoton Light-sheet Microscopy at Optimal Pulse Frequency for Fast In Vivo Imaging

2020 ◽  
Author(s):  
Vincent Maioli ◽  
Antoine Boniface ◽  
Pierre Mahou ◽  
Júlia Ferrer Ortas ◽  
Lamiae Abdeladim ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Light Sheet ◽  
Pulse Frequency ◽  
Light Sheet Microscopy

scholarly journals Genetic suppression of defective profilin by attenuated Myosin II reveals a potential role for Myosin II in actin dynamics in vivo in fission yeast

2020 ◽  
Vol 31 (19) ◽  
pp. 2107-2114 ◽  
Author(s):  
Paola Zambon ◽  
Saravanan Palani ◽  
Shekhar Sanjay Jadhav ◽  
Pananghat Gayathri ◽  
Mohan K. Balasubramanian
Keyword(s):  
Fission Yeast ◽  
Double Mutant ◽  
Potential Role ◽  
Myosin Ii ◽  
Model Organism ◽  
Actin Dynamics ◽  
Mutant Analysis ◽  
Genetic Suppression

This work reveals an in vivo role for Myosin II in actin dynamics, potentially in its disassembly and turnover. The work uses double mutant analysis to arrive at this conclusion using the fission yeast as a model organism.

scholarly journals Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

2017 ◽  
Vol 145 ◽  
pp. S70-S71
Author(s):  
Prameet Kaur ◽  
Timothy E. Saunders ◽  
Nicholas Tolwinski
Keyword(s):  
Signal Transduction ◽  
Light Sheet ◽  
Light Sheet Microscopy

Functional in vivo imaging using fluorescence lifetime light sheet microscopy (Conference Presentation)

2019 ◽  
Author(s):  
Simon M. Ameer-Beg ◽  
Claire A. Mitchell ◽  
Simon P. Poland ◽  
Robert D. Knight ◽  
Guoqing Wang ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
In Vivo Imaging ◽  
Light Sheet ◽  
Light Sheet Microscopy

In vivo light‐sheet microscopy resolves localisation patterns of FSD1 , a superoxide dismutase with function in root development and osmoprotection

2020 ◽  
Vol 44 (1) ◽  
pp. 68-87 ◽  
Author(s):  
Petr Dvořák ◽  
Yuliya Krasylenko ◽  
Miroslav Ovečka ◽  
Jasim Basheer ◽  
Veronika Zapletalová ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Root Development ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Multiview tiling light sheet microscopy for 3D high resolution live imaging

Development ◽  
2021 ◽  
Author(s):  
Mostafa Aakhte ◽  
H.-Arno J. Müller
Keyword(s):  
High Resolution ◽  
Myosin Ii ◽  
Resolution Enhancement ◽  
Light Sheet ◽  
Field Of View ◽  
Large Field ◽  
Axial Resolution ◽  
Light Sheet Microscopy ◽  
Drosophila Embryogenesis ◽  
Mechanical Rotation

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

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