Cortical myosin minifilaments orchestrate the arrangement of microridge protrusions on epithelial cell surfaces

ABSTRACTActin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally-elongated protrusions arranged in maze-like patterns on mucosal epithelial cells that rearrange dynamically by fission and fusion. To characterize how microridges mature and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridges continued to lengthen and microridge alignment became increasingly well ordered. Imaging F-actin and Non-Muscle Myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging revealed that individual cortical NMII minifilaments are tethered to protrusions, often connecting adjacent microridges. NMII minifilaments connecting the ends of microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. Our findings demonstrate that as cells mature, microridges continue to remodel and form an increasingly orderly arrangement through a process orchestrated by cortical NMII contraction.

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Stochastic contraction of myosin minifilaments drives evolution of microridge protrusion patterns in epithelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e21-05-0258 ◽

2021 ◽

pp. mbc.E21-05-0258

Author(s):

Aaron P. van Loon ◽

Ivan S. Erofeev ◽

Andrew B. Goryachev ◽

Alvaro Sagasti

Keyword(s):

High Resolution ◽

Epithelial Cells ◽

Myosin Ii ◽

High Resolution Imaging ◽

Cell Surfaces ◽

Initial Development ◽

Zebrafish Larvae ◽

Resolution Imaging ◽

Muscle Myosin

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally-elongated protrusions on mucosal epithelial cells, where they form evenly spaced, maze-like patterns that dynamically remodel by fission and fusion. To characterize how microridges form their highly ordered, subcellular patterns and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridge spacing and alignment became increasingly well ordered. Imaging F-actin and Non-Muscle Myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked fission and fusion rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging allowed us to image individual NMII minifilaments in the apical cortex of cells in live animals, revealing that minifilaments are tethered to protrusions and often connect adjacent microridges. NMII minifilaments connecting the ends of two microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. These findings demonstrate that as cells mature, cortical NMII activity orchestrates a remodeling process that creates an increasingly orderly microridge arrangement. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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An automated high-resolution in vivo screen in zebrafish to identify chemical regulators of myelination

eLife ◽

10.7554/elife.35136 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 31

Author(s):

Jason J Early ◽

Katy LH Cole ◽

Jill M Williamson ◽

Matthew Swire ◽

Hari Kamadurai ◽

...

Keyword(s):

High Resolution ◽

High Resolution Imaging ◽

Analysis Pipeline ◽

Zebrafish Larvae ◽

Spinning Disk ◽

Resolution Imaging ◽

And Function ◽

In Vivo Screen ◽

Broad Interest

Myelinating oligodendrocytes are essential for central nervous system (CNS) formation and function. Their disruption is implicated in numerous neurodevelopmental, neuropsychiatric and neurodegenerative disorders. However, recent studies have indicated that oligodendrocytes may be tractable for treatment of disease. In recent years, zebrafish have become well established for the study of myelinating oligodendrocyte biology and drug discovery in vivo. Here, by automating the delivery of zebrafish larvae to a spinning disk confocal microscope, we were able to automate high-resolution imaging of myelinating oligodendrocytes in vivo. From there, we developed an image analysis pipeline that facilitated a screen of compounds with epigenetic and post-translational targets for their effects on regulating myelinating oligodendrocyte number. This screen identified novel compounds that strongly promote myelinating oligodendrocyte formation in vivo. Our imaging platform and analysis pipeline is flexible and can be employed for high-resolution imaging-based screens of broad interest using zebrafish.

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An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Journal of Microscopy ◽

10.1111/jmi.12682 ◽

2018 ◽

Vol 270 (3) ◽

pp. 290-301 ◽

Cited By ~ 3

Author(s):

J.E. MALAMY ◽

M. SHRIBAK

Keyword(s):

Wound Healing ◽

Cell Migration ◽

High Resolution ◽

Epithelial Cell ◽

High Resolution Imaging ◽

Epithelial Cell Migration ◽

Resolution Imaging

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Alternative approaches to ultra-high resolution imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087562 ◽

1991 ◽

Vol 49 ◽

pp. 650-651

Author(s):

J.M. Cowley

Keyword(s):

High Resolution ◽

High Voltage ◽

High Resolution Electron Microscopy ◽

Crystal Defects ◽

High Resolution Imaging ◽

General Applicability ◽

Resolution Electron ◽

Radiation Resistant ◽

Resolution Imaging ◽

Alternative Approaches

By extrapolation of past experience, it would seem that the future of ultra-high resolution electron microscopy rests with the advances of electron optical engineering that are improving the instrumental stability of high voltage microscopes to achieve the theoretical resolutions of 1Å or better at 1MeV or higher energies. While these high voltage instruments will undoubtedly produce valuable results on chosen specimens, their general applicability has been questioned on the basis of the excessive radiation damage effects which may significantly modify the detailed structures of crystal defects within even the most radiation resistant materials in a period of a few seconds. Other considerations such as those of cost and convenience of use add to the inducement to consider seriously the possibilities for alternative approaches to the achievement of comparable resolutions.

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Principle and practice of high-resolution imaging with a field-emission TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121090 ◽

1992 ◽

Vol 50 (1) ◽

pp. 138-139

Author(s):

Max T. Otten ◽

Wim M.J. Coene

Keyword(s):

High Resolution ◽

Field Emission ◽

Incidence Angle ◽

Temporal Coherence ◽

Energy Spread ◽

High Resolution Imaging ◽

Major Improvement ◽

High Resolution Images ◽

Resolution Imaging

High-resolution imaging with a LaB6 instrument is limited by the spatial and temporal coherence, with little contrast remaining beyond the point resolution. A Field Emission Gun (FEG) reduces the incidence angle by a factor 5 to 10 and the energy spread by 2 to 3. Since the incidence angle is the dominant limitation for LaB6 the FEG provides a major improvement in contrast transfer, reducing the information limit to roughly one half of the point resolution. The strong improvement, predicted from high-resolution theory, can be seen readily in diffractograms (Fig. 1) and high-resolution images (Fig. 2). Even if the information in the image is limited deliberately to the point resolution by using an objective aperture, the improved contrast transfer close to the point resolution (Fig. 1) is already worthwhile.

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Imaging of ferroelectric domain walls by electron holography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121636 ◽

1992 ◽

Vol 50 (1) ◽

pp. 246-247

Author(s):

Xiao Zhang

Keyword(s):

Magnetic Field ◽

High Resolution ◽

Domain Walls ◽

Ferroelectric Domain ◽

Object Wave ◽

Electron Holography ◽

High Resolution Imaging ◽

Quantitative Measurements ◽

Resolution Imaging ◽

Electron Microscopes

Electron holography has recently been available to modern electron microscopy labs with the development of field emission electron microscopes. The unique advantage of recording both amplitude and phase of the object wave makes electron holography a effective tool to study electron optical phase objects. The visibility of the phase shifts of the object wave makes it possible to directly image the distributions of an electric or a magnetic field at high resolution. This work presents preliminary results of first high resolution imaging of ferroelectric domain walls by electron holography in BaTiO3 and quantitative measurements of electrostatic field distribution across domain walls.

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The method of replication affects metal film granularity and resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155517 ◽

1989 ◽

Vol 47 ◽

pp. 708-709

Author(s):

George C. Ruben

Keyword(s):

Electron Beam ◽

High Resolution ◽

Film Thickness ◽

Single Molecule ◽

Metal Film ◽

Vertical Surface ◽

High Resolution Imaging ◽

Controllable Factors ◽

Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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Performance Evaluation of a Novel Type of Low-Energy Electron Microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180525 ◽

1990 ◽

Vol 48 (1) ◽

pp. 354-355

Author(s):

Bertholdand Senftinger ◽

Helmut Liebl

Keyword(s):

Electron Microscope ◽

High Resolution ◽

Field Strength ◽

Numerical Aperture ◽

Low Energy ◽

Energy Electron ◽

High Resolution Imaging ◽

Lens System ◽

Resolution Imaging ◽

Low Energy Electron

During the last few years the investigation of clean and adsorbate-covered solid surfaces as well as thin-film growth and molecular dynamics have given rise to a constant demand for high-resolution imaging microscopy with reflected and diffracted low energy electrons as well as photo-electrons. A recent successful implementation of a UHV low-energy electron microscope by Bauer and Telieps encouraged us to construct such a low energy electron microscope (LEEM) for high-resolution imaging incorporating several novel design features, which is described more detailed elsewhere.The constraint of high field strength at the surface required to keep the aberrations caused by the accelerating field small and high UV photon intensity to get an improved signal-to-noise ratio for photoemission led to the design of a tetrode emission lens system capable of also focusing the UV light at the surface through an integrated Schwarzschild-type objective. Fig. 1 shows an axial section of the emission lens in the LEEM with sample (28) and part of the sample holder (29). The integrated mirror objective (50a, 50b) is used for visual in situ microscopic observation of the sample as well as for UV illumination. The electron optical components and the sample with accelerating field followed by an einzel lens form a tetrode system. In order to keep the field strength high, the sample is separated from the first element of the einzel lens by only 1.6 mm. With a numerical aperture of 0.5 for the Schwarzschild objective the orifice in the first element of the einzel lens has to be about 3.0 mm in diameter. Considering the much smaller distance to the sample one can expect intense distortions of the accelerating field in front of the sample. Because the achievable lateral resolution depends mainly on the quality of the first imaging step, careful investigation of the aberrations caused by the emission lens system had to be done in order to avoid sacrificing high lateral resolution for larger numerical aperture.

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