In dieser Arbeit wurde eine Methode entwickelt, mit deren Hilfe simultan das Schwimmverhalten und... more In dieser Arbeit wurde eine Methode entwickelt, mit deren Hilfe simultan das Schwimmverhalten und die intrazelluläre Ca 2+-Konzentration von schwimmenden Spermien des Seeigels A. punctulata und des Seesterns A. amurensis beobachtet und analysiert wurden. Es konnte gezeigt werden, dass sowohl die extrazelluläre Stimulation von A. punctulata Spermien mit dem Lockstoff Resact als auch die intrazelluläre Freisetzung von cGMP eine Folge von Ca 2+-Spikes im Flagellum auslösen. Ein Ca 2+-Spike löst eine kombinierte Verhaltensantwort aus. Diese Verhaltensantwort besteht aus einer Biegung in der Schwimmtrajektorie gefolgt von einem Abschnitt geraden Schwimmens ("turn and run"). Die Beobachtung der intrazellulären Ca 2+-Konzentration von schwimmenden Spermienin einem definierten Lockstoff-Gradienten zeigte, dass die induzierten Ca 2+-Spikes und die Stimulus-Funktion synchronisiert sind. Daraus lässt sich schlussfolgern, dass die zeitliche Abfolge von Ca 2+-Spikes das Schwimmverhalten steuert. Anhand der Ergebnisse dieser Arbeit wurde ein Modell zur Chemotaxis von Spermien erstellt, das erläutert wie Spermien sich in einem Gradienten orientieren. Das Eizellpeptid des Seesterns A. amurensis Asterosap wurde als Lockstoff identifieziert. Der Vergleich der Chemotaxis zwischen Spermien des Seeigels A. punctulata und des Seesterns A. amurensis zeigte, dass das chemotaktische Verhalten beider Spezies in Bezug auf 1. die cGMP-induzierte Ca 2+-Dynamik im Flagellum, 2. die durch Ca 2+ ausgelöste Verhaltensantwort und 3. das Schwimmverhalten in einem Lockstoff-Gradienten vergleichbar ist. Die Schlussfolgerung aus diesem Vergleich ist, dass cAMP weder bei der Chemotaxis von Spermien des Seesterns noch von Spermien des Seeigels eine essentielle Rolle spielt. Der cGMP-vermittelte chemotaktische Signalweg ist demnach in zwei Spezies konserviert geblieben, die vor ca. 500 Millionen Jahren divergierten. Ein cGMP-aktivierter Ionenkanal, der für eine Hyperpolarisation oder für einen Ca 2+-Einstrom verantwortlich sein könnte, konnte in A. punctulata bisher nicht identifiziert werden. Womöglich spielen spannungsabhängige Ca 2+-Kanäle, die bisher nur mit der akrosomalen Exozytose in Verbindung gebracht wurden, doch eine bedeutsame Rolle für den Ca 2+-Einsrom in der Chemotaxis von Spermien.
The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cel... more The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-b in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Fö rster resonance energy transfer experiments using dual-fluorophore-labeled Importin-b. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.
The events that occur during chemotaxis of sperm are only partly known. As an essential step towa... more The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca 2 þ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca 2 þ spikes in the flagellum. A Ca 2 þ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-andrun'). The train of Ca 2 þ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca 2 þ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca 2 þ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca 2 þ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.
The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cycl... more The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca 2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca 2+-regulated proteins that-in mature sperm-are involved in flagellar bending.
Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebra... more Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca 2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis 1-3. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined. In particular, the ion channels that are involved in Ca 2+ influx and their mechanisms of gating are not known 4. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K +-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca 2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca 2+ entry indicates the involvement of low-voltage-activated Ca v channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Ca v channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors.
and troponin T) indicates that mutant cardiomyocytes like cells also exhibited a progressive diso... more and troponin T) indicates that mutant cardiomyocytes like cells also exhibited a progressive disorganization of their contractile apparatus. This cellular model describes for the first time a complex morphological pathology caused by the dysfunction of an ion channel. Finally, the study of current proton leak in physiological conditions provides helpful understanding on its pathological impact.
Protein diffusion in lipid membranes is a key aspect of many cellular signaling processes. To qua... more Protein diffusion in lipid membranes is a key aspect of many cellular signaling processes. To quantitatively describe protein diffusion in membranes, several competing theoretical models have been proposed. Among these, the Saffman-Delbrü ck model is the most famous. This model predicts a logarithmic dependence of a protein's diffusion coefficient on its inverse hydrodynamic radius (D f ln 1/R) for small radius values. For large radius values, it converges toward a D f 1/R scaling. Recently, however, experimental data indicate a Stokes-Einstein-like behavior (D f 1/R) of membrane protein diffusion at small protein radii. In this study, we investigate protein diffusion in black lipid membranes using dual-focus fluorescence correlation spectroscopy. This technique yields highly accurate diffusion coefficients for lipid and protein diffusion in membranes. We find that despite its simplicity, the Saffman-Delbrü ck model is able to describe protein diffusion extremely well and a Stokes-Einstein-like behavior can be ruled out.
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Papers by Qui Pham van