scholarly journals Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

2015 ◽  
Vol 26 (11) ◽  
pp. 2044-2053 ◽  
Author(s):  
Comert Kural ◽  
Ahmet Ata Akatay ◽  
Raphaël Gaudin ◽  
Bi-Chang Chen ◽  
Wesley R. Legant ◽  
...  
Keyword(s):  
Leading Edge ◽  
Light Sheet ◽  
Carcinoma Cells ◽  
Coated Pits ◽  
Membrane Flow ◽  
Light Sheet Microscopy ◽  
Motile Cells ◽  
Proposed Model ◽  
Membrane Protrusions ◽  
U373 Cells

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.

scholarly journals Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

2021 ◽  
Author(s):  
Janine J.G. Arts ◽  
Eike K. Mahlandt ◽  
Max L.B. Grönloh ◽  
Lilian Schimmel ◽  
Ivar Noordstra ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Light Sheet ◽  
Cell Junctions ◽  
Light Sheet Microscopy ◽  
Rac1 Activity ◽  
Membrane Protrusions ◽  
Vessel Walls ◽  
Inflamed Tissue

AbstractUpon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

scholarly journals Combination of photoactivation with lattice light-sheet imaging reveals PA-Rac1 generates untemplated, lamellar ruffles

2020 ◽  
Author(s):  
Finian Leyden ◽  
Sanjeev Uthishtran ◽  
U K Moorthi ◽  
H M York ◽  
A Patil ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dorsal Surface ◽  
Experimental System ◽  
Light Sheet ◽  
Small Gtpase ◽  
Local Concentration ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Membrane Protrusions ◽  
A Cell

ABSTRACTMembrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles.

scholarly journals A tent pole twist on membrane ruffles

2018 ◽  
Vol 217 (11) ◽  
pp. 3774-3775
Author(s):  
Reuben V. Philip ◽  
Rene E. Harrison
Keyword(s):  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Cell Biol ◽  
Membrane Protrusions ◽  
Membrane Ruffle

Macropinocytosis or “cell drinking” involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy, Condon et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201804137) reveal the presence of parallel membrane protrusions termed “tent poles” that flank and direct membrane ruffle formation.

scholarly journals An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

2019 ◽  
Vol 30 (17) ◽  
pp. 2254-2267 ◽  
Author(s):  
James J. Faust ◽  
Arnat Balabiyev ◽  
John M. Heddleston ◽  
Nataly P. Podolnikova ◽  
D. Page Baluch ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Cytochalasin B ◽  
Inflammatory Diseases ◽  
Leading Edge ◽  
Light Sheet ◽  
Giant Cells ◽  
Light Sheet Microscopy ◽  
Syndrome Protein

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

scholarly journals An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

2019 ◽  
Author(s):  
James J. Faust ◽  
Arnat Balabiyev ◽  
John M. Heddleston ◽  
Nataly P. Podolnikova ◽  
D. Page Baluch ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Cytochalasin B ◽  
Inflammatory Diseases ◽  
Leading Edge ◽  
Light Sheet ◽  
Giant Cells ◽  
Light Sheet Microscopy ◽  
Syndrome Protein

AbstractMacrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 ± 1 μm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 ± 7 μm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actin-based protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

scholarly journals Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Janine JG Arts ◽  
Eike K Mahlandt ◽  
Max Grönloh ◽  
Lilian Schimmel ◽  
Ivar Noordstra ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Light Sheet ◽  
Cell Junctions ◽  
Light Sheet Microscopy ◽  
Rac1 Activity ◽  
Membrane Protrusions ◽  
Vessel Walls ◽  
Inflamed Tissue

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

scholarly journals Rac1 activation can generate untemplated, lamellar membrane ruffles

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
F. Leyden ◽  
S. Uthishtran ◽  
U. K. Moorthi ◽  
H. M. York ◽  
A. Patil ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dorsal Surface ◽  
Experimental System ◽  
Light Sheet ◽  
Small Gtpase ◽  
Complex Nature ◽  
Local Concentration ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Membrane Protrusions

Abstract Background Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Results Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles. Conclusions Combining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

scholarly journals Achromatic terahertz Airy beam generation with dielectric metasurfaces

Nanophotonics ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Qingqing Cheng ◽  
Juncheng Wang ◽  
Ling Ma ◽  
Zhixiong Shen ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Biomedical Imaging ◽  
Frequency Dispersion ◽  
Light Sheet ◽  
Photonic Devices ◽  
Self Healing ◽  
Beam Focusing ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Potential Applications ◽  
Airy Beams

AbstractAiry beams exhibit intriguing properties such as nonspreading, self-bending, and self-healing and have attracted considerable recent interest because of their many potential applications in photonics, such as to beam focusing, light-sheet microscopy, and biomedical imaging. However, previous approaches to generate Airy beams using photonic structures have suffered from severe chromatic problems arising from strong frequency dispersion of the scatterers. Here, we design and fabricate a metasurface composed of silicon posts for the frequency range 0.4–0.8 THz in transmission mode, and we experimentally demonstrate achromatic Airy beams exhibiting autofocusing properties. We further show numerically that a generated achromatic Airy-beam-based metalens exhibits self-healing properties that are immune to scattering by particles and that it also possesses a larger depth of focus than a traditional metalens. Our results pave the way to the realization of flat photonic devices for applications to noninvasive biomedical imaging and light-sheet microscopy, and we provide a numerical demonstration of a device protocol.

scholarly journals Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

2021 ◽  
Vol 93 (8) ◽  
pp. 4092-4099
Author(s):  
Bing Li ◽  
Aleks Ponjavic ◽  
Wei-Hsin Chen ◽  
Lee Hopkins ◽  
Craig Hughes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Light Sheet Microscopy
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