Abstract. Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spokedeficient mutants (pfl4, pfl7) was increased to wildtype level after treatment with the kinase-inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N~-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 #M cAMP to spokeless axonemes, reconstituted with RU, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutam strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKItreated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.YNEINS are a family of microtubule-based mechanoenzymes responsible for eukaryotic ciliary and flagellar motility, as well as for certain forms of cytoplasmic transport (Gibbons, 1989). Although much is known about the composition and structural organization of dyneins in flagellar axonemes (Witman, 1992), little is known about the coordinated regulation of dynein-driven microtubule sliding that produces regular bending of flagella (see Brokaw, 1994).
Diel migrations of the golden shiner (Notemigonus crysoleucas) from the littoral to limnetic zone of a small Michigan lake were documented through visual observations and gill netting. During the day golden shiners schooled in the littoral zone. Just after sunset schools broke up and the golden shiner migrated to the open water regions of the lake. Feeding by the golden shiner was largely coincident with the evening migration. The majority of the shiner population fed on Daphnia and most prey were obtained within 1 h of sunset. Feeding was minimal through the night with some feeding evident at dawn. Daphnia underwent a strong diel vertical migration, especially larger individuals, and showed a strong horizontal gradient of decreasing size and density from the limnetic into the littoral region. Size of Daphnia eaten by the shiner increased dramatically across the evening feeding period. This increase is due to the horizontal gradient in Daphnia and availability of larger Daphnia as they migrate to the surface. On only one date when prey densities were highest did the shiners actively select large Daphnia. The golden shiner faces a dynamic prey system both in time and space which influences prey "selection." We relate adaptations of planktivorous fishes that migrate to relative abundances of fishes in small lakes. Key words: coevolution, Daphnia, diel migration, fish, foraging behavior, golden shiner, Notemigonus, planktivore, predator–prey
The structure and stability of single- and double-stranded DNA hybrids immobilized on gold are strongly affected by nucleotide-surface interactions. To systematically analyze the effects of these interactions, a set of model DNA hybrids was prepared in conformations that ranged from end-tethered double-stranded to directly adsorbed single-stranded (hairpins) and characterized by surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The stabilities of these hybrids were evaluated by exposure to a series of stringency rinses in solutions of successively lower ionic strength and by competitive hybridization experiments. In all cases, directly adsorbed DNA hybrids are found to be significantly less stable than either free or end-tethered hybrids. The surface-induced weakening and the associated asymmetry in hybridization responses of the two strands forming hairpin stems are most pronounced for single-stranded hairpins containing blocks of m adenine (A) nucleotides and n thymine (T) nucleotides, which have high and low affinity for gold surfaces, respectively. The results allow a qualitative scale of relative stabilities to be developed for DNA hybrids on surfaces. Additionally, the results suggest a route for selectively weakening portions of immobilized DNA hybrids and for introducing asymmetric hybridization responses by using sequence design to control nucleotide-surface interactions--a strategy that may be used in advanced biosensors and in switches or other active elements in DNA-based nanotechnology.
Because cell biology has rapidly increased in breadth and depth, instructors are challenged not only to provide undergraduate science students with a strong, up-to-date foundation of knowledge, but also to engage them in the scientific process. To these ends, revision of the Cell Biology Lab course at the University of Wisconsin-La Crosse was undertaken to allow student involvement in experimental design, emphasize data collection and analysis, make connections to the ''big picture,'' and increase student interest in the field. Multiweek laboratory modules were developed as a method to establish an inquiry-based learning environment. Each module utilizes relevant techniques to investigate one or more questions within the context of a fictional story, and there is a progression during the semester from more instructor-guided to more open-ended student investigation. An assessment tool was developed to evaluate student attitudes regarding their lab experience. Analysis of five semesters of data strongly supports the module format as a successful model for inquiry education by increasing student interest and improving attitude toward learning. In addition, student performance on inquiry-based assignments improved over the course of each semester, suggesting an improvement in inquiry-related skills.
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