Leah T. Haimo

Bidirectional pigment granule movements of melanophores are regulated by protein phosphorylation and dephosphorylation.

Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy.

Microtubules and Microtubule Motors: Mechanisms of Regulation

Microtubule-based motility is precisely regulated, and the targets of regulation may be the motor proteins, the microtubules, or both components of this intricately controlled system. Regulation of microtubule behavior can be mediated by cell cycle-dependent changes in centrosomal microtubule nucleating ability and by cell-specific, microtubule-associated proteins (MAPs). Changes in microtubule organization and dynamics have been correlated with changes in phosphorylation. Regulation of motor proteins may be required both to initiate movement and to dictate its direction. Axonemal and cytoplasmic dyneins as well as kinesin can be phosphorylated and this modification may affect the motor activities of these enzymes or their ability to interact with organelles. A more complete understanding of how motors can be modulated by phosphorylation, either of the motor proteins or of other associated substrates, will be necessary in order to understand how bidirectional transport is regulated.

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesins ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.

Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis

Many motile processes are regulated such that movement occurs only upon activation of a signaling cascade. Sperm from a variety of species are initially quiescent and must be activated prior to beating. The signaling events leading to the activation and regulation of sperm motility are not well characterized. Mature seminal vesicle sperm from the water strider Aquarius remigis are immotile in vitro, but vigorous motility is activated by trypsin. Trypsin‐activated motility was blocked by pretreatment of the sperm with BAPTA‐AM to chelate intracellular Ca2+ and was partially rescued by subsequent addition of A23187 and Ca2+. Thapsigargin stimulated motility in the absence of trypsin, suggesting that intracellular Ca2+ stores are available. In addition, motility could be fully activated by the phosphatase inhibitor calyculin A, suggesting that the immotile state is maintained by an endogenous phosphatase and that kinase activity is required for motility. The MEK1/2 inhibitor U0126 significantly reduced trypsin activated motility, and MPM‐2, an antibody which recognizes proline‐directed phosphorylation by kinases such as MAPK, recognized components of the water strider sperm flagellum. Antibodies specific for the mouse protease activated receptor PAR2 recognized an antigen on the sperm flagellum. These results suggest that trypsin stimulates a Ca2+ and MAPK mediated signaling pathway and potentially implicate a PAR2‐like protein in regulating motility.

Waveform Generation Is Controlled by Phosphorylation and Swimming Direction Is Controlled by Ca2+ in Sperm from the Mosquito Culex quinquefasciatus

ABSTRACT Most animal sperm are quiescent in the male reproductive tract and become activated after mixing with accessory secretions from the male and/or female reproductive tract. Sperm from the mosquito Culex quinquefasciatus initiate flagellar motility after mixing with male accessory gland components, and the sperm flagellum displays three distinct motility patterns over time: a low amplitude, a long wavelength form (Wave A), a double waveform consisting of two superimposed waveforms over the length of the flagellum (Wave B), and finally, a single helical waveform that propels the sperm at high velocity (Wave C). This flagellar behavior is replicated by treating quiescent sperm with trypsin. When exposed to either broad spectrum or tyrosine kinase inhibitors, sperm activated by accessory gland secretions exhibited motility through Wave B but were unable to progress to Wave C. The MEK1/2 inhibitor UO126 and the ERK1/2 inhibitor FR180204 each blocked the transition from Wave B to Wave C, indicating a role for MAPK activity in the control of waveform and, accordingly, progressive movement. Furthermore, a MAPK substrate antibody stained the flagellum of activated sperm. In the absence of extracellular Ca2+, a small fraction of sperm swam backwards, whereas most could not be activated by either accessory glands or trypsin and were immotile. However, the phosphatase inhibitor okadaic acid in the absence of extracellular Ca2+ induced all sperm to swim backwards with a flagellar waveform similar to Wave A. These results indicate that flagellar waveform generation and direction of motility are controlled by protein phosphorylation and Ca2+ levels, respectively.

Germ-cell hub position in a heteropteran testis correlates with the sequence and location of spermatogenesis and production of elaborate sperm bundles.

In insects, spermatogonial cells undergo several mitotic divisions with incomplete cytokinesis, and then proceed through meiosis and spermatogenesis in synchrony. The cells derived from a single spermatogonial cell are referred to as a cyst. In the water strider Aquarius remigis, spermiogenesis occurs within two bi‐lobed testes. In contrast to most insects, in which the germ‐cell hub is located apically and sequential stages of spermatogenesis can be seen moving toward the base of the testis, each lobe of the water strider testis contains a single germ‐cell hub located medially opposite to the efferent duct of the lobe; the developing cysts are displaced toward the distal ends of the lobe as spermiogenesis proceeds. Water strider sperm have both a long flagellum and an unusually long acrosome. The water strider spermatids elongate most of the flagellum prior to morphogenesis of the acrosome, and exhibit several stages of nuclear remodeling before the final, mature sperm nucleus is formed. The maturing sperm are aligned in register in the cyst, and the flagella fold into a coiled bundle while their acrosomes form a rigid helical process that extends from the cyst toward the efferent duct. Mol. Reprod. Dev. 82: 295–304, 2015.