Lukas Kapitein - Academia.edu (original) (raw)

Papers by Lukas Kapitein

Nature Communications, 2015

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleto... more Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure.

Nature, Jan 5, 2005

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bip... more During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separatio...

Frontiers in Neuroanatomy, 2014

Dendritic spines are micron-sized protrusions that harbor the majority of excitatory synapses in ... more Dendritic spines are micron-sized protrusions that harbor the majority of excitatory synapses in the central nervous system. The head of the spine is connected to the dendritic shaft by a 50-400 nm thin membrane tube, called the spine neck, which has been hypothesized to confine biochemical and electric signals within the spine compartment. Such compartmentalization could minimize interspinal crosstalk and thereby support spine-specific synapse plasticity. However, to what extent compartmentalization is governed by spine morphology, and in particular the diameter of the spine neck, has remained unresolved. Here, we review recent advances in tool developmentboth experimental and theoretical -that facilitate studying the role of the spine neck in compartmentalization. Special emphasis is given to recent advances in microscopy methods and quantitative modeling applications as we discuss compartmentalization of biochemical signals, membrane receptors and electrical signals in spines. Multidisciplinary approaches should help to answer how dendritic spine architecture affects the cellular and molecular processes required for synapse maintenance and modulation.

Methods in Cell Biology, 2010

Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, th... more Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, the major sites of excitatory synaptic input. By imaging microtubules in cultured mature primary hippocampal neurons using fluorescently tagged tubulin and microtubule plus-end binding (EB) protein EB3, dynamic microtubules were found to regularly depart from the dendritic shaft and enter dendritic spines. Evidence indicates that microtubule invasions into spines regulate spine actin dynamics and induce transient morphological changes, such as the formation of spine head protrusion and spine growth. Because alterations in spine morphology play an important role in synaptic plasticity and have been linked to learning and memory formation, it is possible that dynamic microtubules are engaged in adaptive processes in the adult brain. This chapter provides detailed methods for live imaging of dynamic microtubules in mature hippocampal neurons in culture. We describe protocols for culturing and transfecting mature hippocampal neurons and visualizing microtubules and microtubule plus-EB proteins by total internal reflection fluorescence microscopy and spinning disk confocal microscopy.

Nature, 2015

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events s... more Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

Nature Chemical Biology, 2006

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell divis... more Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division 4 , represent alternatives to antimitotic agents that target tubulin 5,6 . While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol 1 , act through poorly understood allosteric mechanisms, not competing with ATP binding 7,8 . Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays 9 . We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany, 2008

ABSTRACT Motor proteins move cargos in cells and are involved in coordinated large-scale force ge... more ABSTRACT Motor proteins move cargos in cells and are involved in coordinated large-scale force generation e.g. in cell locomotion, cell division or muscle contraction. Optical traps in conjunction with high-resolution light microscopy has been a tool of choice to analyze dynamic properties of motors, such as power strokes, step lengths or unitary forces. Much detail about microscopic function has also been learned by direct imaging.

PLoS Biology, 2010

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and pr... more The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.

The Journal of Physical Chemistry B, 2004

To obtain high-resolution information on position or conformation of a molecule and at the same t... more To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.

Journal of Neuroscience, 2011

Dynamic microtubules are important to maintain neuronal morphology and function, but whether neur... more Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.

The Journal of Cell Biology, 2008

The EMBO Journal, 2010

Membrane and secretory trafficking are essential for proper neuronal development. However, the mo... more Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

Science, 2011

Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spin... more Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents via various cellular mechanisms, including dampening of transforming growth factor-β signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.

Science, 2010

This copy is for your personal, non-commercial use only.

Review of Scientific Instruments, 2003

High-resolution force and displacement measurements by laser interferometry, combined with optica... more High-resolution force and displacement measurements by laser interferometry, combined with optical tweezers in a light microscope, are frequently based on near-infrared lasers. With common silicon PN photodiodes the bandwidth of detection was found to be limited to about 5 kHz at 1064 nm laser wavelength. This is caused by the fact that silicon becomes increasingly transparent for wavelengths approaching the

Review of Scientific Instruments, 2003

Nature, 2005

Real-time PCR quantification of reverse transcripts NL4-3 HSA viral stocks prepared by transfecti... more Real-time PCR quantification of reverse transcripts NL4-3 HSA viral stocks prepared by transfection of 293T cells were treated with 60 U ml 21 of Turbo DNase I (Ambion) for 1 h at 37 8C to remove residual plasmid DNA. Cells (0.5 £ 10 6 ) were infected as described above, and total DNA was prepared at the indicated time points and stored in 100 ml of 1 mM Tris, pH 7.4. Early and late HIV-1 reverse transcription products were quantified using specific primer and probe combinations. Reaction mixtures (25 ml) contained QuantiTect Probe PCR master mix (Qiagen), 300 nM primers, 200 nM probe and 5 ml of total DNA. PCR was performed for 15 min at 95 8C followed by 40 cycles of 15 s at 95 8C, and 1 min at 60 8C using an ABI Prism 7700 (Applied Biosystems). The specific amplification of newly synthesized reverse transcripts was monitored by treatment with 3TC.

Molecular and Cellular Neuroscience, 2011

To establish and maintain their polarized morphology, neurons employ active transport driven by c... more To establish and maintain their polarized morphology, neurons employ active transport driven by cytoskeletal motor proteins to sort cargo between axons and dendrites. These motors can move in a specific direction over either microtubules (kinesins, dynein) or actin filaments (myosins). The basic traffic rules governing polarized transport on the neuronal cytoskeleton have long remained unclear, but recent work has revealed several fundamental sorting principles based on differences in the cytoskeletal organization in axons versus dendrites. We will highlight the basic characteristics of the neuronal cytoskeleton and review existing evidence for microtubule and actin based traffic rules in polarized neuronal transport. We will propose a model in which polarized sorting of cargo is established by recruiting or activating the proper subset of motor proteins, which are subsequently guided to specific directions by the polarized organization of the neuronal cytoskeleton.

Journal of Molecular Biology, 2010

Both Grand Unified symmetries and discrete flavour symmetries are appealing ways to describe appa... more Both Grand Unified symmetries and discrete flavour symmetries are appealing ways to describe apparent structures in the gauge and flavour sectors of the Standard Model. Both symmetries put constraints on the high energy behaviour of the theory. This can give rise to unexpected interplay when building models that possess both symmetries. We investigate on the possibility to combine a Pati-Salam model with the discrete flavour symmetry S 4 that gives rise to quark-lepton complementarity. Under appropriate assumptions at the GUT scale, the model reproduces fermion masses and mixings both in the quark and in the lepton sectors. We show that in particular the Higgs sector and the running Yukawa couplings are strongly affected by the combined constraints of the Grand Unified and family symmetries. This in turn reduces the phenomenologically viable parameter space, with high energy mass scales confined to a small region and some parameters in the neutrino sector slightly unnatural. In the allowed regions, we can reproduce the quark masses and the CKM matrix. In the lepton sector, we reproduce the charged lepton masses, including bottom-tau unification and the Georgi-Jarlskog relation as well as the two known angles of the PMNS matrix. The neutrino mass spectrum can present a normal or an inverse hierarchy, and only allowing the neutrino parameters to spread into a range of values between λ −2 and λ 2 , with λ ≃ 0.2. Finally, our model suggests that the reactor mixing angle is close to its current experimental bound.

Current Biology, 2014

Proper organization of microtubule networks depends on microtubule-associated proteins and motors... more Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1-3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends [4]. To explore the mechanics of kinesin-guided microtubule growth, we reconstituted this process in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90°, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90°, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. Our findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays.

Nature Communications, 2015

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleto... more Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure.

Nature, Jan 5, 2005

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bip... more During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separatio...

Frontiers in Neuroanatomy, 2014

Dendritic spines are micron-sized protrusions that harbor the majority of excitatory synapses in ... more Dendritic spines are micron-sized protrusions that harbor the majority of excitatory synapses in the central nervous system. The head of the spine is connected to the dendritic shaft by a 50-400 nm thin membrane tube, called the spine neck, which has been hypothesized to confine biochemical and electric signals within the spine compartment. Such compartmentalization could minimize interspinal crosstalk and thereby support spine-specific synapse plasticity. However, to what extent compartmentalization is governed by spine morphology, and in particular the diameter of the spine neck, has remained unresolved. Here, we review recent advances in tool developmentboth experimental and theoretical -that facilitate studying the role of the spine neck in compartmentalization. Special emphasis is given to recent advances in microscopy methods and quantitative modeling applications as we discuss compartmentalization of biochemical signals, membrane receptors and electrical signals in spines. Multidisciplinary approaches should help to answer how dendritic spine architecture affects the cellular and molecular processes required for synapse maintenance and modulation.

Methods in Cell Biology, 2010

Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, th... more Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, the major sites of excitatory synaptic input. By imaging microtubules in cultured mature primary hippocampal neurons using fluorescently tagged tubulin and microtubule plus-end binding (EB) protein EB3, dynamic microtubules were found to regularly depart from the dendritic shaft and enter dendritic spines. Evidence indicates that microtubule invasions into spines regulate spine actin dynamics and induce transient morphological changes, such as the formation of spine head protrusion and spine growth. Because alterations in spine morphology play an important role in synaptic plasticity and have been linked to learning and memory formation, it is possible that dynamic microtubules are engaged in adaptive processes in the adult brain. This chapter provides detailed methods for live imaging of dynamic microtubules in mature hippocampal neurons in culture. We describe protocols for culturing and transfecting mature hippocampal neurons and visualizing microtubules and microtubule plus-EB proteins by total internal reflection fluorescence microscopy and spinning disk confocal microscopy.

Nature, 2015

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events s... more Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

Nature Chemical Biology, 2006

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell divis... more Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division 4 , represent alternatives to antimitotic agents that target tubulin 5,6 . While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol 1 , act through poorly understood allosteric mechanisms, not competing with ATP binding 7,8 . Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays 9 . We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany, 2008

ABSTRACT Motor proteins move cargos in cells and are involved in coordinated large-scale force ge... more ABSTRACT Motor proteins move cargos in cells and are involved in coordinated large-scale force generation e.g. in cell locomotion, cell division or muscle contraction. Optical traps in conjunction with high-resolution light microscopy has been a tool of choice to analyze dynamic properties of motors, such as power strokes, step lengths or unitary forces. Much detail about microscopic function has also been learned by direct imaging.

PLoS Biology, 2010

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and pr... more The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.

The Journal of Physical Chemistry B, 2004

To obtain high-resolution information on position or conformation of a molecule and at the same t... more To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.

Journal of Neuroscience, 2011

Dynamic microtubules are important to maintain neuronal morphology and function, but whether neur... more Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.

The Journal of Cell Biology, 2008

The EMBO Journal, 2010

Membrane and secretory trafficking are essential for proper neuronal development. However, the mo... more Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

Science, 2011

Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spin... more Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents via various cellular mechanisms, including dampening of transforming growth factor-β signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.

Science, 2010

This copy is for your personal, non-commercial use only.

Review of Scientific Instruments, 2003

High-resolution force and displacement measurements by laser interferometry, combined with optica... more High-resolution force and displacement measurements by laser interferometry, combined with optical tweezers in a light microscope, are frequently based on near-infrared lasers. With common silicon PN photodiodes the bandwidth of detection was found to be limited to about 5 kHz at 1064 nm laser wavelength. This is caused by the fact that silicon becomes increasingly transparent for wavelengths approaching the

Review of Scientific Instruments, 2003

Nature, 2005

Real-time PCR quantification of reverse transcripts NL4-3 HSA viral stocks prepared by transfecti... more Real-time PCR quantification of reverse transcripts NL4-3 HSA viral stocks prepared by transfection of 293T cells were treated with 60 U ml 21 of Turbo DNase I (Ambion) for 1 h at 37 8C to remove residual plasmid DNA. Cells (0.5 £ 10 6 ) were infected as described above, and total DNA was prepared at the indicated time points and stored in 100 ml of 1 mM Tris, pH 7.4. Early and late HIV-1 reverse transcription products were quantified using specific primer and probe combinations. Reaction mixtures (25 ml) contained QuantiTect Probe PCR master mix (Qiagen), 300 nM primers, 200 nM probe and 5 ml of total DNA. PCR was performed for 15 min at 95 8C followed by 40 cycles of 15 s at 95 8C, and 1 min at 60 8C using an ABI Prism 7700 (Applied Biosystems). The specific amplification of newly synthesized reverse transcripts was monitored by treatment with 3TC.

Molecular and Cellular Neuroscience, 2011

To establish and maintain their polarized morphology, neurons employ active transport driven by c... more To establish and maintain their polarized morphology, neurons employ active transport driven by cytoskeletal motor proteins to sort cargo between axons and dendrites. These motors can move in a specific direction over either microtubules (kinesins, dynein) or actin filaments (myosins). The basic traffic rules governing polarized transport on the neuronal cytoskeleton have long remained unclear, but recent work has revealed several fundamental sorting principles based on differences in the cytoskeletal organization in axons versus dendrites. We will highlight the basic characteristics of the neuronal cytoskeleton and review existing evidence for microtubule and actin based traffic rules in polarized neuronal transport. We will propose a model in which polarized sorting of cargo is established by recruiting or activating the proper subset of motor proteins, which are subsequently guided to specific directions by the polarized organization of the neuronal cytoskeleton.

Journal of Molecular Biology, 2010

Both Grand Unified symmetries and discrete flavour symmetries are appealing ways to describe appa... more Both Grand Unified symmetries and discrete flavour symmetries are appealing ways to describe apparent structures in the gauge and flavour sectors of the Standard Model. Both symmetries put constraints on the high energy behaviour of the theory. This can give rise to unexpected interplay when building models that possess both symmetries. We investigate on the possibility to combine a Pati-Salam model with the discrete flavour symmetry S 4 that gives rise to quark-lepton complementarity. Under appropriate assumptions at the GUT scale, the model reproduces fermion masses and mixings both in the quark and in the lepton sectors. We show that in particular the Higgs sector and the running Yukawa couplings are strongly affected by the combined constraints of the Grand Unified and family symmetries. This in turn reduces the phenomenologically viable parameter space, with high energy mass scales confined to a small region and some parameters in the neutrino sector slightly unnatural. In the allowed regions, we can reproduce the quark masses and the CKM matrix. In the lepton sector, we reproduce the charged lepton masses, including bottom-tau unification and the Georgi-Jarlskog relation as well as the two known angles of the PMNS matrix. The neutrino mass spectrum can present a normal or an inverse hierarchy, and only allowing the neutrino parameters to spread into a range of values between λ −2 and λ 2 , with λ ≃ 0.2. Finally, our model suggests that the reactor mixing angle is close to its current experimental bound.

Current Biology, 2014

Proper organization of microtubule networks depends on microtubule-associated proteins and motors... more Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1-3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends [4]. To explore the mechanics of kinesin-guided microtubule growth, we reconstituted this process in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90°, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90°, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. Our findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays.