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Larisa Gheber - One of the best experts on this subject based on the ideXlab platform.
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drag induced Directionality switching of kinesin 5 cin8 revealed by cluster motility analysis
2021Co-Authors: Himanshu Pandey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of Directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic Directionality. The underlying mechanism, however, remains unexplained. Here, we studied clustering-induced Directionality switching of the bidirectional kinesin-5 Cin8. Based on the characterization of single-molecule and cluster motility, we developed a model that predicts that Directionality switching of Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag. The model shows excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. Our analysis identifies a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to seemingly unrelated experimental factors including changes in motor density and molecular crowding, and in multimotor motility assays.
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drag induced Directionality switching of kinesin 5 cin8 revealed by cluster motility analysis
2020Co-Authors: Himanshu Handey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of Directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic Directionality. While several kinesin motors have now been shown to exhibit context-dependent Directionality that can be altered under diverse experimental conditions, the underlying mechanism remains unknown. Here, we studied clustering-induced Directionality switching of the mitotic kinesin-5 Cin8, using a fluorescence-based single-molecule motility assay combined with biophysical theory. Based on the detailed characterization of the motility of single motors and clusters of Cin8, we developed a predictive molecular model, that quantitatively agrees with experimental data. This combined approach allowed us to quantify the response of Cin8 motors to external forces as well as the interactions between Cin8 motors, and thereby develop a detailed understanding of the molecular mechanism underlying Directionality switching. The main insight is that Directionality switching is caused by a single feature of Cin8: an asymmetric response of active motion to forces that oppose motion, here referred to as drag. This general mechanism explains why bidirectional motor proteins are capable of reversing direction in response to seemingly unrelated experimental factors including clustering, changes in the ionic strength of the buffer, increased motor density and molecular crowding, and in motility assays.
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regulation and possible physiological role of bi directional motility of the kinesin 5 cin8
2016Co-Authors: Ofer Shapira, Jawdat Albassam, Alina Goldstein, Larisa GheberAbstract:The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch Directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this Directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear.Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and Directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its Directionality from fast minus- to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end Directionality.
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Directionality of individual kinesin 5 cin8 motors is modulated by loop 8 ionic strength and microtubule geometry
2011Co-Authors: Adina Gersongurwitz, Christina Thiede, Natalia Movshovich, Vladimir Fridman, Maria Podolskaya, Tsafi Danieli, Stefan Lakamper, Dieter R Klopfenstein, Christoph F Schmidt, Larisa GheberAbstract:Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch Directionality. Here, we have examined Directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of Directionality control for the anaphase function of Cin8.
Jakob Christensendalsgaard - One of the best experts on this subject based on the ideXlab platform.
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Directionality of auditory nerve fiber responses to pure tone stimuli in the grassfrog rana temporaria ii spike timing
1997Co-Authors: Morten Buhl Jorgensen, Jakob ChristensendalsgaardAbstract:We studied the Directionality of spike timing in the responses of single auditory nerve fibers of the grass frog, Rana temporaria, to tone burst stimulation. Both the latency of the first spike after stimulus onset and the preferred firing phase during the stimulus were studied. In addition, the Directionality of the phase of eardrum vibrations was measured. The response latency showed systematic and statistically significant changes with sound direction at both low and high frequencies. The latency changes were correlated with response strength (spike rate) changes and were probably the result of directional changes in effective stimulus intensity. Systematic changes in the preferred firing phase were seen in all fibers that showed phaselocking (i.e., at frequencies below 500–700 Hz). The mean phase lead for stimulation from the contralateral side was approximately 140° at 200 Hz and decreased to approximately 100° at 700 Hz. These phaseshifts correspond to differences in spike timing of approximately 2 ms and 0.4 ms respectively. The phaseshifts were nearly independent of stimulus intensity. The phase Directionality of eardrum vibrations was smaller than that of the nerve fibers. Hence, the strong directional phaseshifts shown by the nerve fibers probably reflect the directional characteristics of extratympanic pathways.
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Directionality of auditory nerve fiber responses to pure tone stimuli in the grassfrog rana temporaria i spike rate responses
1997Co-Authors: Morten Buhl Jorgensen, Jakob ChristensendalsgaardAbstract:We studied the Directionality of spike rate responses of auditory nerve fibers of the grassfrog, Rana temporaria, to pure tone stimuli. All auditory fibers showed spike rate Directionality. The strongest Directionality was seen at low frequencies (200-400 Hz), where the spike rate could change by up to nearly 200 spikes s-1, with sound direction. At higher frequencies the directional spike rate changes were mostly below 100 spikes s-1. In equivalent dB SPL terms (calculated using the fibers' rate-intensity curves) the maximum directionalities were up to 15 dB at low frequencies and below 10 dB at higher frequencies. Two types of directional patterns were observed. At frequencies below 500 Hz relatively strong responses were evoked by stimuli from the ipsilateral (+90 degrees) and contralateral (-90 degrees) directions while the weakest responses were evoked by stimuli from frontal (0 degree or +30 degrees) or posterior (-135 degrees) directions. At frequencies above 800 Hz the strongest responses were evoked by stimuli from the ipsilateral direction while gradually weaker responses were seen as the sound direction shifted towards the contralateral side. At frequencies between 500 and 800 Hz both directional patterns were seen. The Directionality was highly intensity dependent. No special adaptations for localization of conspecific calls were found.
Morten Buhl Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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Directionality of auditory nerve fiber responses to pure tone stimuli in the grassfrog rana temporaria ii spike timing
1997Co-Authors: Morten Buhl Jorgensen, Jakob ChristensendalsgaardAbstract:We studied the Directionality of spike timing in the responses of single auditory nerve fibers of the grass frog, Rana temporaria, to tone burst stimulation. Both the latency of the first spike after stimulus onset and the preferred firing phase during the stimulus were studied. In addition, the Directionality of the phase of eardrum vibrations was measured. The response latency showed systematic and statistically significant changes with sound direction at both low and high frequencies. The latency changes were correlated with response strength (spike rate) changes and were probably the result of directional changes in effective stimulus intensity. Systematic changes in the preferred firing phase were seen in all fibers that showed phaselocking (i.e., at frequencies below 500–700 Hz). The mean phase lead for stimulation from the contralateral side was approximately 140° at 200 Hz and decreased to approximately 100° at 700 Hz. These phaseshifts correspond to differences in spike timing of approximately 2 ms and 0.4 ms respectively. The phaseshifts were nearly independent of stimulus intensity. The phase Directionality of eardrum vibrations was smaller than that of the nerve fibers. Hence, the strong directional phaseshifts shown by the nerve fibers probably reflect the directional characteristics of extratympanic pathways.
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Directionality of auditory nerve fiber responses to pure tone stimuli in the grassfrog rana temporaria i spike rate responses
1997Co-Authors: Morten Buhl Jorgensen, Jakob ChristensendalsgaardAbstract:We studied the Directionality of spike rate responses of auditory nerve fibers of the grassfrog, Rana temporaria, to pure tone stimuli. All auditory fibers showed spike rate Directionality. The strongest Directionality was seen at low frequencies (200-400 Hz), where the spike rate could change by up to nearly 200 spikes s-1, with sound direction. At higher frequencies the directional spike rate changes were mostly below 100 spikes s-1. In equivalent dB SPL terms (calculated using the fibers' rate-intensity curves) the maximum directionalities were up to 15 dB at low frequencies and below 10 dB at higher frequencies. Two types of directional patterns were observed. At frequencies below 500 Hz relatively strong responses were evoked by stimuli from the ipsilateral (+90 degrees) and contralateral (-90 degrees) directions while the weakest responses were evoked by stimuli from frontal (0 degree or +30 degrees) or posterior (-135 degrees) directions. At frequencies above 800 Hz the strongest responses were evoked by stimuli from the ipsilateral direction while gradually weaker responses were seen as the sound direction shifted towards the contralateral side. At frequencies between 500 and 800 Hz both directional patterns were seen. The Directionality was highly intensity dependent. No special adaptations for localization of conspecific calls were found.
Brian Vohnsen - One of the best experts on this subject based on the ideXlab platform.
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analysing the impact of myopia on the stiles crawford effect of the first kind using a digital micromirror device
2018Co-Authors: Alessandra Carmichael Martins, Brian VohnsenAbstract:PURPOSE: Photoreceptor light acceptance is closely tied to the Stiles-Crawford effect of the first kind (SCE-I). Whether the SCE-I plays a role in myopic development remains unclear although a reduction in Directionality has been predicted for high myopia. The purpose of this study is to analyse the relationship between foveal SCE-I Directionality, axial eye length, and defocus for emmetropic subjects wearing ophthalmic trial lenses during psychophysical measurements and for myopic subjects with their natural correction. METHOD: A novel uniaxial flicker system has been implemented making use of a Digital Micromirror Device (DMD) to flicker between a 2.3 visual degrees circular reference and a set of circular test patterns in a monocular Maxwellian view at 0.5 Hz. The brightness of the test is adjusted by the duty cycle of the projected light to an upper limit of 22 727 Hz. The wavelength and bandwidth are set by a tuneable liquid-crystal filter centred at 550 nm. A total of four measurement series for 11 pupil entrance points have been realized for the right eye of 6 emmetropic and 10 myopic subjects whose pupils were dilated with tropicamide. Five of the emmetropic subjects wore ophthalmic trial lenses in the range of -3 to +9 dioptres to mimic hyperopic to highly myopic vision and resulting visibility plots have been fitted to a Gaussian SCE-I function. In turn, the myopic subjects wore their natural correction during the analysis of the SCE-I. All subjects had their axial eye length determined with an ultrasound device. RESULTS: A SCE-I Directionality parameter in the range of 0.03 to 0.06/mm2 was found for the emmetropic subjects with corrected vision in fair agreement to values in the literature. The results also revealed a marked reduction in Directionality in the range from 16% to 30% with every 3 dioptre increase of simulated myopia, as well as a 10% increased Directionality in simulated hyperopic eyes. For both emmetropic and myopic subjects, a decrease in Directionality with increase in axial length was found in agreement with theoretical expectations. CONCLUSION: The study confirms a clear link between SCE-I Directionality, uncorrected defocus, and axial eye length. This may play a role for emmetropization and thus myopic progression as cone photoreceptors capture light from a wider pupil area in elongated eyes due to a geometrical scaling.
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simulating human photoreceptor optics using a liquid filled photonic crystal fiber
2011Co-Authors: Diego Rativa, Brian VohnsenAbstract:We introduce a liquid-filled photonic crystal fiber to simulate a retinal cone photoreceptor mosaic and the Directionality selective mechanism broadly known as the Stiles-Crawford effect. Experimental measurements are realized across the visible spectrum to study waveguide coupling and Directionality at different managed waveguide parameters. The crystal fiber method is a hybrid tool between theory and a real biological sample and a valuable addition as a retina model for real eye simulations.
Jawdat Albassam - One of the best experts on this subject based on the ideXlab platform.
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drag induced Directionality switching of kinesin 5 cin8 revealed by cluster motility analysis
2021Co-Authors: Himanshu Pandey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of Directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic Directionality. The underlying mechanism, however, remains unexplained. Here, we studied clustering-induced Directionality switching of the bidirectional kinesin-5 Cin8. Based on the characterization of single-molecule and cluster motility, we developed a model that predicts that Directionality switching of Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag. The model shows excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. Our analysis identifies a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to seemingly unrelated experimental factors including changes in motor density and molecular crowding, and in multimotor motility assays.
-
drag induced Directionality switching of kinesin 5 cin8 revealed by cluster motility analysis
2020Co-Authors: Himanshu Handey, Jawdat Albassam, Emanuel Reithmann, Alina Goldsteinlevitin, Erwin Frey, Larisa GheberAbstract:Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of Directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic Directionality. While several kinesin motors have now been shown to exhibit context-dependent Directionality that can be altered under diverse experimental conditions, the underlying mechanism remains unknown. Here, we studied clustering-induced Directionality switching of the mitotic kinesin-5 Cin8, using a fluorescence-based single-molecule motility assay combined with biophysical theory. Based on the detailed characterization of the motility of single motors and clusters of Cin8, we developed a predictive molecular model, that quantitatively agrees with experimental data. This combined approach allowed us to quantify the response of Cin8 motors to external forces as well as the interactions between Cin8 motors, and thereby develop a detailed understanding of the molecular mechanism underlying Directionality switching. The main insight is that Directionality switching is caused by a single feature of Cin8: an asymmetric response of active motion to forces that oppose motion, here referred to as drag. This general mechanism explains why bidirectional motor proteins are capable of reversing direction in response to seemingly unrelated experimental factors including clustering, changes in the ionic strength of the buffer, increased motor density and molecular crowding, and in motility assays.
-
regulation and possible physiological role of bi directional motility of the kinesin 5 cin8
2016Co-Authors: Ofer Shapira, Jawdat Albassam, Alina Goldstein, Larisa GheberAbstract:The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch Directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this Directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear.Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and Directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its Directionality from fast minus- to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end Directionality.
