Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa

Pigment organelles known as melanosomes disperse or aggregate in a melanophore in response to hormones. These movements are mediated by the microtubule motors kinesin-2 and cytoplasmic dynein. However, the force generation mechanism of dynein, unlike that of kinesin, is not well understood. In this study, to address this issue, we investigated the dynein-mediated aggregation of melanosomes in zebrafish melanophores. We applied the fluctuation theorem of non-equilibrium statistical mechanics to estimate forces acting on melanosomes during transport by dynein, given that the energy of a system is related to its fluctuation. Our results demonstrate that multiple force-producing units cooperatively transport a single melanosome. Since the force is generated by dynein, this suggests that multiple dyneins carry a single melanosome. Cooperative transport has been reported for other organelles; thus, multiple-motor transport may be a universal mechanism for moving organelles within the cell.

Section: Resultsmentioning

confidence: 83%

Investigation of multiple-dynein transport of melanosomes by non-invasive force measurement using fluctuation unit χ

Hasegawa

Sagawa

Ikeda

et al. 2019

“…Ciliobrevin A (HPI-4, SIGMA-ALDRICH) was diluted in dimethyl sulfoxide (DMSO, SIGMA-ALDRICH) at a concentration of 10 mM and stored at −20 °C. Live cell microscopy experiments were performed at a ciliobrevin A concentration of 50 µM 50 . Before the experiments, the culture medium was removed from the cells, the ciliobrevin A diluted in image medium was added and the cells were incubated at 37 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Phagosomal transport depends strongly on phagosome size

Keller

Berghoff

Kress

2017

Macrophages internalize pathogens for intracellular degradation. An important part of this process is the phagosomal transport from the cell periphery to the perinuclear region. Biochemical factors are known to influence the fate of phagosomes. Here, we show that the size of phagosomes also has a strong influence on their transport. We found that large phagosomes are transported persistently to the nucleus, whereas small phagosomes show strong bidirectional transport. We show that dynein motors play a larger role in the transport of large phagosomes, whereas actin filament-based motility plays a larger role in the transport of small phagosomes. Furthermore, we investigated the spatial distribution of dyneins and microtubules around phagosomes and hypothesize that dynein and microtubule density differences between the nucleus-facing side of phagosomes and the opposite side could explain part of the observed transport characteristics. Our findings suggest that a size-dependent cellular sorting mechanism might exist that supports macrophages in their immunological roles.

“…The sperm suspension was further diluted an additional 100× with F-NSW (filtered natural sea water) containing 0.01% (w/v) Bovine Serum Albumin (Wako, Japan) and placed between a glass slide and a coverslip with two strips of plastic tape (Scotch, 3 M) as spacers. The frequency of flagellar beating was determined by a method described 29 , using a dark-field microscope (Olympus CH; A40 objective lens) equipped with stroboscopic illumination (modified from the original design of 30 ).…”

Section: Methodsmentioning

confidence: 99%

Sperm lacking Bindin are infertile but are otherwise indistinguishable from wildtype sperm

Wessel

Wada

Yajima

et al. 2021

Self Cite

Cell–cell fusion is limited to only a few cell types in the body of most organisms and sperm and eggs are paradigmatic in this process. The specialized cellular mechanism of fertilization includes the timely exposure of gamete–specific interaction proteins by the sperm as it approaches the egg. Bindin in sea urchin sperm is one such gamete interaction protein and it enables species–specific interaction with a homotypic egg. We recently showed that Bindin is essential for fertilization by use of Cas9 targeted gene inactivation in the sea urchin, Hemicentrotus pulcherrimus. Here we show phenotypic details of Bindin-minus sperm. Sperm lacking Bindin do not bind to nor fertilize eggs at even high concentrations, yet they otherwise have wildtype morphology and function. These features include head shape, tail length and beating frequency, an acrosomal vesicle, a nuclear fossa, and they undergo an acrosomal reaction. The only phenotypic differences between wildtype and Bindin-minus sperm identified is that Bindin-minus sperm have a slightly shorter head, likely as a result of an acrosome lacking Bindin. These data, and the observation that Bindin-minus embryos develop normally and metamorphose into normal functioning adults, support the contention that Bindin functions are limited to species–specific sperm–egg interactions. We conclude that the evolutionary divergence of Bindin is not constrained by any other biological roles.