ASSOCIATION OF A MYOSIN MOTOR WITH MEMBRANE-BOUNDED PIGMENT GRANULES IN FRESHWATER SHRIMP CHROMATOPHORES: EVIDENCE FROM THE NITELLA ACTIN-CABLE ASSAY

We have adapted an actin-mosin motility assay to examine the interactions in vitro between actin cables isolated from the giant internodal cells of the freshwater alga, Nitella, and pigment granules extracted from red ovarian chromatophores of the freshwater palaemonid shrimp, Macrobrachium olfersi. The chromatophore pigment mass consists of large (0.5-1.0-mu m diameter) membrane-bounded granules, and small (140-nm diameter), a membranous granules, both structurally continuous with the abundant smooth endoplasmic reticulum. Our previous immunocytochemical studies show a myosin motor to be stably associated with the pigment mass; however, to which granule type or membrane the myosin motor is attached is unclear. Here, we show that sodium vanadate, a myosin ATPase inhibitor, markedly increases the affinity of isolated, large, membrane-bounded granules for Nitella actin cables to which they become permanently attached. This interaction does not occur in granule preparations containing ATP with uninhibited, active myosin without vanadate. We propose that a stable state of elevated affinity is established between the granule-located myosin motor and the Nitella actin cables, resulting from a vanadate-inhibited acto-myosin-ADP complex. This finding provides further evidence for a myosin motor positioned on the surface of the membrane-bounded pigment granules in shrimp ovarian chromatophores.

Signal Transduction, Plasma Membrane Calcium Movements, and Pigment Translocation in Freshwater Shrimp Chromatophores

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by omega-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation. J. Exp. Zool. 313A:605-617, 2010. (C) 2010 Wiley-Liss, Inc.

Cyclic Guanosine Monophosphate Signaling Cascade Mediates Pigment Aggregation in Freshwater Shrimp Chromatophores

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 mu mol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course (approximate to 20 min) and peak velocity (approximate to 17 mu m/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 X 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 mu mol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 mu mol l(-1)) induced moderate aggregation by themselves (approximate to 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 mu mol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 mu mol l(-1)) partially inhibited RPCH-triggered aggregation by approximate to 35%. Escherichia coli heat-stable enterotoxin (STa, 1 mu mol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.

Morphology and biology of a new Pseudopolydora (Annelida: Spionidae) species from Brazil

Adults of Pseudopolydora rosebelae sp. nov. inhabit silty tubes on muddy bottoms in shallow water in southern Brazil, states of Sao Paulo and Rio de Janeiro. They are rare and extremely delicate, attaining 20 mm long for 55 chaetigers. The worms are distinctive by their colourful yellow and black pigmentation on the anterior part of body and palps, prominent transverse hood on the dorsal anterior edge of chaetiger 3, and lack of coloured respiratory pigment in blood. Of 12 examined individuals, all were females. Oogenesis is intraovarian; oocytes develop from chaetigers 14-15 to chaetigers 24-36. Recently laid oocytes were about 150 mu m in diameter, with embryos and developing larvae found in capsules inside female tubes in March-June. Broods comprised up to 23 capsules with 400 propagules. Capsules were joined to each other in a string and each attached by a single thin stalk to the inner wall of the tube. Larvae hatched at the 4-chaetiger stage and fed on plankton. Pelagic larvae are unique among Pseudopolydora in having large ramified mid-dorsal melanophores from chaetiger 3 onwards. Competent larvae are able to settle and metamorphose at the 15-chaetiger stage, but can remain planktonic up to 18 chaetigers. They have one pair of unpigmented ocelli and three pairs of black eyes in the prostomium, unpaired ramified mid-dorsal melanophores on chaetiger 1 and on the pygidium, ramified lateral melanophores on chaetigers 5-10, prominent yellow chromatophores in the prostomium, peristomium, on dorsal and ventral sides of chaetigers and in the pygidium. Branchiae are present on chaetigers 7-10, and gastrotrochs are arranged on chaetigers 3, 5, 7 and 12. Provisional serrated bristles are present in all notopodia, and hooks are present in neuropodia from chaetiger 8 onwards. Two pairs of provisional protonephridia are present in chaetigers 1 and 2, and adult metanephridia are present from chaetiger 4.