Three-dimensional actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

AbstractLeukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 μm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Download Full-text

Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽

10.7554/elife.26990 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 46

Author(s):

Lillian K Fritz-Laylin ◽

Megan Riel-Mehan ◽

Bi-Chang Chen ◽

Samuel J Lord ◽

Thomas D Goddard ◽

...

Keyword(s):

Three Dimensional ◽

Light Sheet ◽

Time Lapse ◽

Three Dimensions ◽

Cortical Actin ◽

Linear Arrangement ◽

Collagen Matrices ◽

Flat Surfaces ◽

Actin Networks ◽

Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Download Full-text

Digital Spindle: A New Way to Explore Mitotic Functions by Whole Cell Data Collection and a Computational Approach

Cells ◽

10.3390/cells9051255 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1255

Author(s):

Norio Yamashita ◽

Masahiko Morita ◽

Hideo Yokota ◽

Yuko Mimori-Kiyosue

Keyword(s):

Cell Biology ◽

Information Science ◽

Three Dimensional ◽

Light Sheet ◽

Live Imaging ◽

Three Dimensions ◽

Complex Data ◽

Spatiotemporal Resolution ◽

Whole Cell ◽

Light Sheet Microscopy

From cells to organisms, every living system is three-dimensional (3D), but the performance of fluorescence microscopy has been largely limited when attempting to obtain an overview of systems’ dynamic processes in three dimensions. Recently, advanced light-sheet illumination technologies, allowing drastic improvement in spatial discrimination, volumetric imaging times, and phototoxicity/photobleaching, have been making live imaging to collect precise and reliable 3D information increasingly feasible. In particular, lattice light-sheet microscopy (LLSM), using an ultrathin light-sheet, enables whole-cell 3D live imaging of cellular processes, including mitosis, at unprecedented spatiotemporal resolution for extended periods of time. This technology produces immense and complex data, including a significant amount of information, raising new challenges for big image data analysis and new possibilities for data utilization. Once the data are digitally archived in a computer, the data can be reused for various purposes by anyone at any time. Such an information science approach has the potential to revolutionize the use of bioimage data, and provides an alternative method for cell biology research in a data-driven manner. In this article, we introduce examples of analyzing digital mitotic spindles and discuss future perspectives in cell biology.

Download Full-text

Imaging mitotic processes in three dimensions with lattice light-sheet microscopy

Chromosome Research ◽

10.1007/s10577-021-09656-3 ◽

2021 ◽

Author(s):

Yuko Mimori-Kiyosue

Keyword(s):

Dimensional Space ◽

Three Dimensional ◽

Image Data ◽

Light Sheet ◽

Three Dimensions ◽

Cell Level ◽

Spatiotemporal Resolution ◽

New Era ◽

Light Sheet Microscopy ◽

Three Dimensional Space

AbstractThere are few technologies that can capture mitotic processes occurring in three-dimensional space with the desired spatiotemporal resolution. Due to such technical limitations, our understanding of mitosis, which has been studied since the early 1880s, is still incomplete with regard to mitotic processes and their regulatory mechanisms at a molecular level. A recently developed high-resolution type of light-sheet microscopy, lattice light-sheet microscopy (LLSM), has achieved unprecedented spatiotemporal resolution scans of intracellular spaces at the whole-cell level. This technology enables experiments that were not possible before (e.g., tracking of growth of every spindle microtubule end and discrimination of individual chromosomes in living cells), thus providing a new avenue for the analysis of mitotic processes. Herein, principles of LLSM technology are introduced, as well as experimental techniques that became possible with LLSM. In addition, issues remaining to be solved for use of this technology in mitosis research, big image data problems, are presented to help guide mitosis research into a new era.

Download Full-text

Multi-color 4D superresolution light-sheet microscopy reveals organelle interactions at isotropic 100-nm resolution and sub-second timescales

10.1101/2021.05.09.443230 ◽

2021 ◽

Author(s):

Peng Fei

Keyword(s):

Dimensional Space ◽

Three Dimensional ◽

Light Sheet ◽

Three Dimensions ◽

Live Cells ◽

Spatiotemporal Resolution ◽

Volumetric Imaging ◽

Light Sheet Microscopy ◽

Intracellular Organelles ◽

Microscopy Techniques

Long-term visualization of the dynamic organelle-organelle or protein-organelle interactions throughout the three-dimensional space of whole live cells is essential to better understand their functions, but this task remains challenging due to the limitations of existing three-dimensional fluorescence microscopy techniques, such as an insufficient axial resolution, low volumetric imaging rate, and photobleaching. Here, we present the combination of a progressive deep-learning superresolution strategy with a dual-ring-modulated SPIM design capable of visualizing the dynamics of intracellular organelles in live cells for hours at an isotropic spatial resolution of ~100 nm in three dimensions and a temporal resolution up to ~17 Hz. With a compelling spatiotemporal resolution, we substantially reveal the complex spatial relationships and interactions between the endoplasmic reticulum (ER) and mitochondria throughout live cells, providing new insights into ER-mediated mitochondrial division. We also localized the motion of Drp1 oligomers in three dimensions and observed Drp1-mediated mitochondrial branching for the first time.

Download Full-text

The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System

Micromachines ◽

10.3390/mi10090599 ◽

2019 ◽

Vol 10 (9) ◽

pp. 599 ◽

Cited By ~ 2

Author(s):

Chen ◽

Liu ◽

Lu ◽

Lee ◽

Tsai ◽

...

Keyword(s):

Cell Culture ◽

Hippocampal Neurons ◽

Three Dimensional ◽

Light Sheet ◽

Three Dimensions ◽

Laser Exposure ◽

Spatiotemporal Resolution ◽

Strong Light ◽

Light Sheet Microscopy ◽

Volumetric Images

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Download Full-text

Mechanosensitivity of amoeboid cells crawling in 3D

10.1101/2021.05.11.443058 ◽

2021 ◽

Author(s):

Florian Gaertner ◽

Patricia Reis-Rodrigues ◽

Ingrid de Vries ◽

Miroslav Hons ◽

Juan Aguilera ◽

...

Keyword(s):

Three Dimensional ◽

The Body ◽

Three Dimensions ◽

Cortical Actin ◽

Actin Networks ◽

Motile Cells ◽

Spatio Temporal ◽

Adhesive Interactions ◽

The Right ◽

Amoeboid Cells

Efficient immune-responses require migrating leukocytes to be in the right place at the right time. When crawling through the body amoeboid leukocytes must traverse complex three-dimensional tissue-landscapes obstructed by extracellular matrix and other cells, raising the question how motile cells adapt to mechanical loads to overcome these obstacles. Here we reveal the spatio-temporal configuration of cortical actin-networks rendering amoeboid cells mechanosensitive in three-dimensions, independent of adhesive interactions with the microenvironment. In response to compression, Wiskott-Aldrich syndrom protein (WASp) assembles into dot-like structures acting as nucleation sites for actin spikes that in turn push against the external load. High precision targeting of WASp to objects as delicate as collagen fibers allows the cell to locally and instantaneously deform its viscoelastic surrounding in order to generate space for forward locomotion. Such pushing forces are essential for fast and directed leukocyte migration in fibrous and cell-packed tissues such as skin and lymph nodes.

Download Full-text

Three-dimensional quantification of twisting in the Arabidopsis petiole

Journal of Plant Research ◽

10.1007/s10265-021-01291-7 ◽

2021 ◽

Author(s):

Yuta Otsuka ◽

Hirokazu Tsukaya

Keyword(s):

Cross Sections ◽

Three Dimensional ◽

Light Sheet ◽

Underlying Mechanism ◽

Two Dimensions ◽

Observation Angle ◽

Differential Growth ◽

Light Sheet Microscopy ◽

Definition Of ◽

Twisting Angle

AbstractOrganisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

Download Full-text