Faculty Opinions recommendation of Opposite-polarity motors activate one another to trigger cargo transport in live cells.

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.

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Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C

The Journal of Cell Biology ◽

10.1083/jcb.201812170 ◽

2019 ◽

Vol 218 (9) ◽

pp. 2982-3001 ◽

Cited By ~ 10

Author(s):

Agnieszka A. Kendrick ◽

Andrea M. Dickey ◽

William B. Redwine ◽

Phuoc Tien Tran ◽

Laura Pontano Vaites ◽

...

Keyword(s):

Cytoplasmic Dynein ◽

Opposite Polarity ◽

Full Length ◽

Cell Periphery ◽

Long Distance ◽

Short Region ◽

Cargo Transport ◽

In Cells

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the “tail” of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3’s ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors.

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HOOK3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein and KIF1C

10.1101/508887 ◽

2018 ◽

Cited By ~ 2

Author(s):

Agnieszka A. Kendrick ◽

William B. Redwine ◽

Phuoc Tien Tran ◽

Laura Pontano Vaites ◽

Monika Dzieciatkowska ◽

...

Keyword(s):

Cytoplasmic Dynein ◽

Opposite Polarity ◽

Full Length ◽

General Mechanism ◽

Long Distance ◽

Short Region ◽

Cargo Transport ◽

Single Complex ◽

In Cells

AbstractThe unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations support the existence of mechanisms to couple opposite polarity motors: in cells some cargos rapidly switch directions and kinesin motors can be used to localize dynein. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor HOOK3 and the kinesin-3 KIF1C. Here we show that KIF1C and dynein/dynactin can exist in a single complex scaffolded by HOOK3. Full-length HOOK3 binds to and activates dynein/dynactin motility. HOOK3 also binds to a short region in the “tail” of KIF1C, but unlike dynein/dynactin, this interaction does not affect the processive motility of KIF1C. HOOK3 scaffolding allows dynein to transport KIF1C towards the microtubule minus end, and KIF1C to transport dynein towards the microtubule plus end. We propose that linking dynein and kinesin motors by dynein activating adaptors may be a general mechanism to regulate bidirectional motility.

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4-dimensional, high-resolution imaging of living cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122484 ◽

1992 ◽

Vol 50 (1) ◽

pp. 416-417

Author(s):

Shinya Inoué

Keyword(s):

Light Microscope ◽

Living Cells ◽

Live Cells ◽

Video Tape ◽

Active Living ◽

High Resolution Imaging ◽

3 Dimensional ◽

Dynamic Architecture ◽

Resolution Imaging ◽

Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

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Polarity Determination in Sphalerite and Wurtzite Structures

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136106 ◽

1990 ◽

Vol 48 (2) ◽

pp. 500-501

Author(s):

Stuart McKernan ◽

C. Barry Carter

Keyword(s):

Opposite Polarity ◽

Single Domain ◽

Polar Material ◽

Electron Microscopic ◽

Dynamical Interaction ◽

X Ray ◽

Polarity Determination ◽

The Absolute ◽

Microscopic Techniques

The determination of the absolute polarity of a polar material is often crucial to the understanding of the defects which occur in such materials. Several methods exist by which this determination may be performed. In bulk, single-domain specimens, macroscopic techniques may be used, such as the different etching behavior, using the appropriate etchant, of surfaces with opposite polarity. X-ray measurements under conditions where Friedel’s law (which means that the intensity of reflections from planes of opposite polarity are indistinguishable) breaks down can also be used to determine the absolute polarity of bulk, single-domain specimens. On the microscopic scale, and particularly where antiphase boundaries (APBs), which separate regions of opposite polarity exist, electron microscopic techniques must be employed. Two techniques are commonly practised; the first [1], involves the dynamical interaction of hoLz lines which interfere constructively or destructively with the zero order reflection, depending on the crystal polarity. The crystal polarity can therefore be directly deduced from the relative intensity of these interactions.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002440 ◽

1980 ◽

Vol 38 ◽

pp. 552-553

Author(s):

K.I. Pagh ◽

M.R. Adelman

Keyword(s):

Cell Shape ◽

Physarum Polycephalum ◽

Slime Mold ◽

Live Cells ◽

Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

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A Photoactivable Formaldehyde Donor with Fluorescence Monitoring Reveals Threshold to Arrest Cell Migration

10.26434/chemrxiv.11388321.v1 ◽

2019 ◽

Author(s):

Lukas P Smaga ◽

Nicholas W Pino ◽

Gabriela E Ibarra ◽

Vishnu Krishnamurthy ◽

Jefferson Chan

Keyword(s):

Wound Healing ◽

Cell Migration ◽

Fluorescence Enhancement ◽

Biological Effects ◽

Cellular Systems ◽

Live Cells ◽

First Report ◽

Cell Lysate ◽

Intracellular Release ◽

Biological Analytes

Controlled light-mediated delivery of biological analytes enables the investigation of highly reactivity molecules within cellular systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.

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