104 results on '"Lukas C. Kapitein"'
Search Results
2. Cellular cartography: Towards an atlas of the neuronal microtubule cytoskeleton
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Malina K. Iwanski and Lukas C. Kapitein
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microtubules ,neurons ,microtubule stability ,post-translational modifications ,microtubule-associated proteins ,microscopy ,Biology (General) ,QH301-705.5 - Abstract
Microtubules, one of the major components of the cytoskeleton, play a crucial role during many aspects of neuronal development and function, such as neuronal polarization and axon outgrowth. Consequently, the microtubule cytoskeleton has been implicated in many neurodevelopmental and neurodegenerative disorders. The polar nature of microtubules is quintessential for their function, allowing them to serve as tracks for long-distance, directed intracellular transport by kinesin and dynein motors. Most of these motors move exclusively towards either the plus- or minus-end of a microtubule and some have been shown to have a preference for either dynamic or stable microtubules, those bearing a particular post-translational modification or those decorated by a specific microtubule-associated protein. Thus, it becomes important to consider the interplay of these features and their combinatorial effects on transport, as well as how different types of microtubules are organized in the cell. Here, we discuss microtubule subsets in terms of tubulin isotypes, tubulin post-translational modifications, microtubule-associated proteins, microtubule stability or dynamicity, and microtubule orientation. We highlight techniques used to study these features of the microtubule cytoskeleton and, using the information from these studies, try to define the composition, role, and organization of some of these subsets in neurons.
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- 2023
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3. Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains
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Shih-Te Hung, Arnau Llobet Rosell, Daphne Jurriens, Marijn Siemons, Oleg Soloviev, Lukas C. Kapitein, Kristin Grußmayer, Lukas J. Neukomm, Michel Verhaegen, and Carlas Smith
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Super-resolution Microscopy ,localization microscopy ,adaptive optics ,Drosophila ,brain ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.
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- 2022
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4. ER – lysosome contacts at a pre-axonal region regulate axonal lysosome availability
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Nazmiye Özkan, Max Koppers, Inge van Soest, Alexandra van Harten, Daphne Jurriens, Nalan Liv, Judith Klumperman, Lukas C. Kapitein, Casper C. Hoogenraad, and Ginny G. Farías
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Science - Abstract
In neurons and other cells, contacts between organelles regulates function and subcellular organization, but the precise mechanisms and effects are unclear. Here the authors show that endoplasmic reticulum (ER) tubules in the soma of neurons regulate lysosome localization and function by regulating lysosomal fission, suggesting a role for ER – lysosome inter-organelle membrane contact sites in lysosomal axonal availability.
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- 2021
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5. Robust adaptive optics for localization microscopy deep in complex tissue
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Marijn E. Siemons, Naomi A. K. Hanemaaijer, Maarten H. P. Kole, and Lukas C. Kapitein
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Science - Abstract
It is difficult to apply SMLM to complex biological tissues. Here the authors report REALM, Robust and Effective Adaptive Optics in Localisation Microscopy, to improve SMLM in tissue and use this to resolve the organisation of spectrin in the axon initial segment in brain tissue.
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- 2021
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6. Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton
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Adrian Müller-Deku, Joyce C. M. Meiring, Kristina Loy, Yvonne Kraus, Constanze Heise, Rebekkah Bingham, Klara I. Jansen, Xiaoyi Qu, Francesca Bartolini, Lukas C. Kapitein, Anna Akhmanova, Julia Ahlfeld, Dirk Trauner, and Oliver Thorn-Seshold
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Science - Abstract
Light-based modulation of the microtubule (MT) cytoskeleton is an attractive goal for spatiotemporally-resolved MT studies. Here the authors develop a first generation photoswitchable small molecule MT stabiliser based on paclitaxel, allowing optical control over cellular MT dynamics.
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- 2020
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7. WDR47 protects neuronal microtubule minus ends from katanin-mediated severing
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Robin R. Buijs, Jessica J.A. Hummel, Mithila Burute, Xingxiu Pan, Yujie Cao, Riccardo Stucchi, Maarten Altelaar, Anna Akhmanova, Lukas C. Kapitein, and Casper C. Hoogenraad
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neuron ,axon ,dendrite ,microtubule ,centrosome ,WDR47 ,Biology (General) ,QH301-705.5 - Abstract
Summary: Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.
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- 2021
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8. Wnt Signaling Directs Neuronal Polarity and Axonal Growth
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Eliana Stanganello, Eitan Erez Zahavi, Mithila Burute, Jasper Smits, Ingrid Jordens, Madelon M. Maurice, Lukas C. Kapitein, and Casper C. Hoogenraad
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Science - Abstract
Summary: The establishment of neuronal polarity is driven by cytoskeletal remodeling that stabilizes and promotes the growth of a single axon from one of the multiple neurites. The importance of the local microtubule stabilization in this process has been revealed however, the external signals initiating the cytoskeletal rearrangements are not completely understood. In this study, we show that local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth. We found that in the early stages of neuronal polarization, Wnt3a accumulates in one of the neurites of unpolarized cells and thereby could determine axon positioning. Subsequently, Wnt3a localizes to the growing axon, where it activates the canonical Wnt pathway and controls axon positioning and axonal length. We propose a model in which Wnt3a regulates the formation and growth of the axon by activating local intracellular signaling events leading to microtubule remodeling. : Neuroscience; Molecular Neuroscience; Cellular Neuroscience Subject Areas: Neuroscience, Molecular Neuroscience, Cellular Neuroscience
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- 2019
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9. Probing aggrephagy using chemically-induced protein aggregates
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Anne F. J. Janssen, Eugene A. Katrukha, Wendy van Straaten, Pauline Verlhac, Fulvio Reggiori, and Lukas C. Kapitein
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Science - Abstract
Autophagic clearance of aggregates, aggrephagy, is essential for cellular homeostasis but tools to induce and monitor it are limited. Here the authors present a fluorescence-based aggrephagy induction assay to study spatiotemporal dynamics and control mechanisms driving protein aggregate clearance.
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- 2018
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10. APC2 controls dendrite development by promoting microtubule dynamics
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Olga I. Kahn, Philipp Schätzle, Dieudonnée van de Willige, Roderick P. Tas, Feline W. Lindhout, Sybren Portegies, Lukas C. Kapitein, and Casper C. Hoogenraad
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Science - Abstract
Microtubules in dendrites are characterized by mixed polarity orientation. Here, the authors show a role for adenomatous polyposis coli 2 (APC2) in regulating dendrite microtubule dynamics and dendrite development.
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- 2018
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11. Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots
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Eugene A. Katrukha, Marina Mikhaylova, Hugo X. van Brakel, Paul M. van Bergen en Henegouwen, Anna Akhmanova, Casper C. Hoogenraad, and Lukas C. Kapitein
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Science - Abstract
Methods to probe transport dynamics within cells can shed insight into the nature of the cytoplasm. Here the authors develop a method to functionalize and deliver quantum dots intracellularly to show how the cytoskeleton influences non-equilibrium intracellular transport dynamics.
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- 2017
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12. Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture
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Marta Esteves da Silva, Max Adrian, Philipp Schätzle, Joanna Lipka, Takuya Watanabe, Sukhee Cho, Kensuke Futai, Corette J. Wierenga, Lukas C. Kapitein, and Casper C. Hoogenraad
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synaptic plasticity ,intracellular transport ,AMPA receptors ,dendritic spine ,cytoskeleton ,microtubule ,actin ,myosin ,kinesin ,KIF1C ,myosinV ,myosinVI ,Biology (General) ,QH301-705.5 - Abstract
Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs) at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing endosomes. In addition, how the positioning of AMPAR-containing endosomes affects synapse organization and functioning has never been directly explored. Here, we used live-cell imaging in hippocampal neuron cultures to show that intracellular AMPARs are transported in Rab11-positive recycling endosomes, which frequently enter dendritic spines and depend on the microtubule and actin cytoskeleton. By using chemically induced dimerization systems to recruit kinesin (KIF1C) or myosin (MyosinV/VI) motors to Rab11-positive recycling endosomes, we controlled their trafficking and found that induced removal of recycling endosomes from spines decreases surface AMPAR expression and PSD-95 clusters at synapses. Our data suggest a mechanistic link between endosome positioning and postsynaptic structure and composition.
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- 2015
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13. Myosin-V Induces Cargo Immobilization and Clustering at the Axon Initial Segment
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Anne F. J. Janssen, Roderick P. Tas, Petra van Bergeijk, Rosalie Oost, Casper C. Hoogenraad, and Lukas C. Kapitein
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polarized transport ,motor proteins ,myosin-V ,kinesin ,axon initial segment ,motor cooperation ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS) upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS.
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- 2017
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14. An Iterative Frequency-Based Approach for Gelmap Grid Deformation Correction.
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Vincent J. W. Hellebrekers, Josiah B. Passmore, Lukas C. Kapitein, and Ihor Smal
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- 2024
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15. Correction: Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes.
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Casper C. Hoogenraad, Ioana Popa, Kensuke Futai, Emma Martinez-Sanchez, Phebe S. Wulf, Thijs van Vlijmen, Bjorn R. Dortland, Viola Oorschot, Roland Govers, Maria Monti, Albert J. R. Heck, Morgan Sheng, Judith Klumperman, Holger Rehmann, Dick Jaarsma, Lukas C. Kapitein, and Peter van der Sluijs
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Biology (General) ,QH301-705.5 - Published
- 2010
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16. Direct observation of motor protein stepping in living cells using MINFLUX
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Takahiro Deguchi, Malina K. Iwanski, Eva-Maria Schentarra, Christopher Heidebrecht, Lisa Schmidt, Jennifer Heck, Tobias Weihs, Sebastian Schnorrenberg, Philipp Hoess, Sheng Liu, Veronika Chevyreva, Kyung-Min Noh, Lukas C. Kapitein, and Jonas Ries
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Multidisciplinary ,Article - Abstract
Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable futurein vivostudies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.
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- 2023
17. A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle
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Klara I. Jansen, Malina K. Iwanski, Mithila Burute, and Lukas C. Kapitein
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Cell Biology - Abstract
The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.
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- 2023
18. GelMap: Intrinsic calibration and deformation mapping for expansion microscopy
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Hugo G.J. Damstra, Josiah B. Passmore, Albert K. Serweta, Ioannis Koutlas, Mithila Burute, Frank J. Meye, Anna Akhmanova, and Lukas C. Kapitein
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Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.
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- 2022
19. Axonal ER tubules regulate local translation via P180/RRBP1-mediated ribosome interactions
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Max Koppers, Nazmiye Özkan, Ha H. Nguyen, Daphne Jurriens, Janine McCaughey, Riccardo Stucchi, Maarten Altelaar, Lukas C. Kapitein, Casper C. Hoogenraad, and Ginny G. Farias
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SUMMARYLocal mRNA translation in axons is critical for the spatial and temporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons, however how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with ER tubules in the axon in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation in an mRNA-dependent manner. Our results establish an important role for the axonal ER in localizing mRNA translation and in dynamically regulating the axonal proteome in response to neuronal stimuli.
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- 2022
20. Author response: Self-assembly of pericentriolar material in interphase cells lacking centrioles
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Fangrui Chen, Jingchao Wu, Malina K Iwanski, Daphne Jurriens, Arianna Sandron, Milena Pasolli, Gianmarco Puma, Jannes Z Kromhout, Chao Yang, Wilco Nijenhuis, Lukas C Kapitein, Florian Berger, and Anna Akhmanova
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- 2022
21. The SARS-CoV-2 accessory factor ORF7a downregulates MHC class I surface expression
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Shuxuan Zheng, Hendrik de Buhr, Patrique Praest, Anouk Evers, Ingrid Brak-Boer, Mariëlle van Grinsven, Ylenia Longo, Liset de Vries, Wilco Nijenhuis, Lukas C. Kapitein, Jeffrey M. Beekman, Monique Nijhuis, Ingo Drexler, Emmanuel J. H. J. Wiertz, and Robert Jan Lebbink
- Abstract
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19.Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.
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- 2022
22. Increased microtubule lattice spacing correlates with selective binding of kinesin-1 in cells
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Leanne de Jager, Klara I. Jansen, Lukas C. Kapitein, Friedrich Förster, and Stuart C. Howes
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Within the cell cargo is transported via motor proteins walking along microtubules. The affinity of motor proteins for microtubules is controlled by various layers of regulation like tubulin isoforms, post- translational modifications and microtubule associated proteins. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but to what extent it acts as an additional layer of regulation has remained unclear. In this study, we used cryo-correlative light and electron microscopy to study microtubule lattices inside cells. We find that, while most microtubules have a compacted lattice (∼41 Å), a significant proportion of the microtubule cores have expanded lattice spacings and that these lattice spacings could be modulated by the microtubule stabilizing drug Taxol. Furthermore, kinesin-1 predominantly binds microtubules with a more expanded lattice spacing (∼41.6 Å). The different lattice spacings present in the cell can thus act as an additional factor that modulates the binding of motor proteins to specific microtubule subsets.
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- 2022
23. A motor-based approach to induce chromosome-specific mis-segregations in human cells
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My Anh Truong, Paula Cané-Gasull, Sippe G. de Vries, Wilco Nijenhuis, René Wardenaar, Lukas C. Kapitein, Floris Foijer, and Susanne M.A. Lens
- Abstract
Various cancer types exhibit highly characteristic and recurrent aneuploidy patterns. The origin of these cancer type-specific karyotypes, and the extent to which they contribute to cancer progression, remains to be elucidated, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to mis-segregate specific chromosomes at will in different human cell types. We employed Tet repressor (TetR) or nuclease dead Cas9 (dCas9) to link a plant-derived microtubule minus-end-directed kinesin (Physcomitrella patens Kinesin14VIb) to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were often counteracted by forces from kinetochore-attached microtubules. This tug of war resulted in chromosome-specific segregation errors during anaphase, and revealed that spindle forces can heavily stretch chromosomal arms. Using chromosome-specific FISH and single-cell whole genome sequencing, we established that motor-induced mis-segregations result in specific arm-level, and to a lesser extent, whole chromosome aneuploidies, after a single cell division. Our kinesin-based strategy to manipulate individual mitotic chromosomes opens up the possibility to investigate the immediate cellular responses to specific (arm level) aneuploidies in different cell types; an important step towards understanding how recurrent aneuploidy patterns arise in different cancer types.
- Published
- 2022
24. Mechanisms of microtubule organization in differentiated animal cells
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Anna, Akhmanova and Lukas C, Kapitein
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Organelles ,Animals ,Biological Transport ,Cell Differentiation ,Microtubule-Associated Proteins ,Microtubules ,Cytoskeleton - Abstract
Microtubules are polarized cytoskeletal filaments that serve as tracks for intracellular transport and form a scaffold that positions organelles and other cellular components and modulates cell shape and mechanics. In animal cells, the geometry, density and directionality of microtubule networks are major determinants of cellular architecture, polarity and proliferation. In dividing cells, microtubules form bipolar spindles that pull chromosomes apart, whereas in interphase cells, microtubules are organized in a cell type-specific fashion, which strongly correlates with cell physiology. In motile cells, such as fibroblasts and immune cells, microtubules are organized as radial asters, whereas in immotile epithelial and neuronal cells and in muscles, microtubules form parallel or antiparallel arrays and cortical meshworks. Here, we review recent work addressing how the formation of such microtubule networks is driven by the plethora of microtubule regulatory proteins. These include proteins that nucleate or anchor microtubule ends at different cellular structures and those that sever or move microtubules, as well as regulators of microtubule elongation, stability, bundling or modifications. The emerging picture, although still very incomplete, shows a remarkable diversity of cell-specific mechanisms that employ conserved building blocks to adjust microtubule organization in order to facilitate different cellular functions.
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- 2022
25. 3D multi-color far-red single-molecule localization microscopy with probability-based fluorophore classification
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Marijn E. Siemons, Daphne Jurriens, Carlas S. Smith, and Lukas C. Kapitein
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Single-Molecule Localization Microscopy remains limited in its ability for robust and simple multi-color imaging. Whereas the fluorophore Alexa647 is widely used due to its brightness and excellent blinking dynamics, other excellent blinking fluorophores, such as CF660 and CF680, spectrally overlap. Here we present Probability-based Fluorophore Classification, a method to perform multi-color SMLM with Alexa647, CF660 and CF680 that uses statistical decision theory for optimal classification. The emission is split in a short and long wavelength channel to enable classification and localization without a major loss in localization precision. Each emitter is classified using a Generalized Maximum Likelihood Ratio Test using the photon statistics of both channels. This easy-to-adopt approach does not require nanometer channel registration, is able to classify fluorophores with tunable low false positive rates (
- Published
- 2022
26. Author response: Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
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Hugo GJ Damstra, Boaz Mohar, Mark Eddison, Anna Akhmanova, Lukas C Kapitein, and Paul W Tillberg
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- 2022
27. Rücktitelbild: Unravelling Channel Structure–Diffusivity Relationships in Zeolite ZSM‐5 at the Single‐Molecule Level (Angew. Chem. 5/2022)
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Donglong Fu, J. J. Erik Maris, Katarina Stanciakova, Nikolaos Nikolopoulos, Onno Heijden, Laurens D. B. Mandemaker, Marijn E. Siemons, Desiree Salas Pastene, Lukas C. Kapitein, Freddy T. Rabouw, Florian Meirer, and Bert M. Weckhuysen
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General Medicine - Published
- 2021
28. Back Cover: Unravelling Channel Structure–Diffusivity Relationships in Zeolite ZSM‐5 at the Single‐Molecule Level (Angew. Chem. Int. Ed. 5/2022)
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Donglong Fu, J. J. Erik Maris, Katarina Stanciakova, Nikolaos Nikolopoulos, Onno Heijden, Laurens D. B. Mandemaker, Marijn E. Siemons, Desiree Salas Pastene, Lukas C. Kapitein, Freddy T. Rabouw, Florian Meirer, and Bert M. Weckhuysen
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General Chemistry ,Catalysis - Published
- 2021
29. Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells
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Roderick P. Tas, Eelco J.P. de Koning, Françoise Carlotti, Esther de Graaff, Cyntha M. van den Berg, Casper C. Hoogenraad, Sybren Portegies, Fransje W. J. Boot, Bastiaan J. Viergever, Ivar Noordstra, Lukas C. Kapitein, Eugene A. Katrukha, Ka Lou Yu, and Anna Akhmanova
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biology ,Chemistry ,Insulin ,medicine.medical_treatment ,Pancreatic islets ,Integrin ,Fluorescence recovery after photobleaching ,Cell biology ,Extracellular matrix ,Focal adhesion ,medicine.anatomical_structure ,Myosin ,medicine ,biology.protein ,Beta (finance) - Abstract
Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including Bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain non-neuronal proteins LL5β and KANK1, which in migrating cells organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. While previous analyses in vitro and in neurons suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.
- Published
- 2021
30. Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells
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Theun M De Kort, Henriette H. M. Raeven, Lukas C. Kapitein, Marielle M.P. van Grinsven, Robert Jan Lebbink, Malina K. Iwanski, Dorien de Jong, Gimano D. Amatngalim, Hugo G.J. Damstra, Sacha Spelier, Emma J. van Grinsven, Zahra E. Soltani, Patrique Praest, Jesse E. Brunsveld, Monique Nijhuis, Anna Akhmanova, Wilco Nijenhuis, Jeffrey M. Beekman, and Lisa W. Rodenburg
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viruses ,Viral pathogenesis ,STED microscopy ,Biology ,Golgi apparatus ,medicine.disease_cause ,Cell biology ,symbols.namesake ,Viral life cycle ,Viral entry ,medicine ,Fluorescence microscope ,symbols ,Motile cilium ,Coronavirus - Abstract
A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.
- Published
- 2021
31. Matrix elasticity gradients guide neuronal polarity by controlling microtubule network mobility
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Lukas C. Kapitein, Klara I. Jansen, Tina Vermonden, Marko Mihajlovic, and Mithila Burute
- Subjects
medicine.anatomical_structure ,Neurite ,Microtubule ,Chemistry ,medicine ,Extracellular ,Biophysics ,Matrix (biology) ,Axon ,Optogenetics ,Cytoskeleton ,Intracellular - Abstract
Neuronal polarization and axon specification depend on extracellular cues, intracellular signaling, cytoskeletal rearrangements and polarized transport, but the interplay between these processes has remained unresolved. The polarized transport of kinesin-1 into a specific neurite is an early marker for axon identity, but the mechanisms that govern neurite selection and polarized transport are unknown. We show that extracellular elasticity gradients control polarized transport and axon specification, mediated by Rho-GTPases whose local activation is necessary and sufficient for polarized transport. Selective Kinesin-1 accumulation furthermore depends on differences in microtubule network mobility between neurites and local control over this mobility is necessary and sufficient for proper polarization, as shown using optogenetic anchoring of microtubules. Together, these results explain how mechanical cues can instruct polarized transport and axon specification.
- Published
- 2021
32. Author response: Quantitative mapping of dense microtubule arrays in mammalian neurons
- Author
-
Lukas C. Kapitein, Desiree M Salas Pastene, Eugene A. Katrukha, and Daphne Jurriens
- Subjects
Chemistry ,Microtubule ,Cell biology - Published
- 2021
33. Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia
- Author
-
Gimano D. Amatngalim, Cornelis K. van der Ent, Magarida D. Amaral, Harry G.M. Heijerman, Sacha Vrendenbarg, Ellen Aarts, Wilco Nijenhuis, Cornelis M. van Drunen, Henriette H. M. Raeven, Lisa W. Rodenburg, Bente L. Aalbers, Iris A.L. Silva, Jeffrey M. Beekman, Sabine Michel, Karin M. de Winter-de Groot, Jesse E. Brunsveld, Evelien Kruisselbrink, and Lukas C. Kapitein
- Subjects
Alternative methods ,Combination therapy ,biology ,Chemistry ,Spheroid ,Epithelial monolayer ,respiratory system ,medicine.disease ,Cystic fibrosis ,Cystic fibrosis transmembrane conductance regulator ,respiratory tract diseases ,medicine ,biology.protein ,Cancer research ,Airway ,Cftr modulator - Abstract
Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described application of 3D airway organoids in CFTR function assays have not been fully optimal. In this report we therefore describe an alternative method of culturing nasal brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1β and interleukine-1β, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF.
- Published
- 2021
34. A live-cell marker to visualize the dynamics of stable microtubules
- Author
-
Mithila Burute, Klara I. Jansen, and Lukas C. Kapitein
- Subjects
Immunolabeling ,Tubulin ,Cell division ,biology ,Acetylation ,Chemistry ,Microtubule ,Organelle ,biology.protein ,macromolecular substances ,Cytoskeleton ,Function (biology) ,Cell biology - Abstract
The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for post-translational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present a live-cell marker to visualize stable MTs and explore their dynamics. We demonstrate that a rigor mutant of kinesin-1 binds selectively to acetylated MTs without affecting MT organization and organelle transport. These MTs are long-lived, do not depolymerize upon nocadozale-treatment or laser-based severing, and display rich dynamics, including undulation, looping and sliding. This marker will help to explore how different MT subsets contribute to cellular organization and transport.
- Published
- 2021
35. Direct observation of aggregate-triggered selective autophagy
- Author
-
Lukas C. Kapitein, Wilco Nijenhuis, Anne F.J. Janssen, Eugene A. Katrukha, and Giel Korsten
- Subjects
Omegasome ,Selective autophagy ,Autophagosome ,Live cell imaging ,Autophagy ,Cellular homeostasis ,Aggrephagy ,Biology ,Protein aggregation ,Cell biology - Abstract
Degradation of aggregates by selective autophagy is important as damaged proteins may impose a threat to cellular homeostasis. Although the core components of the autophagy machinery are well-characterized, the spatiotemporal regulation of many selective autophagy processes, including aggrephagy, remains largely unexplored. Furthermore, because most live-cell imaging studies have so far focused on starvation-induced autophagy, little is known about the dynamics of aggrephagy. Here, we describe the development and application of the mKeima-PIM assay, which enables live-cell observation of autophagic turnover and degradation of inducible protein aggregates in conjunction with key autophagy players. This allowed us to quantify the relative timing and duration of different steps of aggrephagy and revealed the short-lived nature of the autophagosome. The assay furthermore showed the spatial distribution of omegasome formation, highlighting that autophagy initiation is directly instructed by the cargo. Moreover, we found that nascent autophagosomes mostly remain immobile until acidification occurs. Thus, our assay provides new insights into the spatiotemporal regulation and dynamics of aggrephagy.
- Published
- 2021
36. Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes
- Author
-
Ian Paterson, Carolyn A. Moores, Tianyang Liu, Ankit Rai, J. Fernando Díaz, Juan Estévez-Gallego, Anna Akhmanova, Eugene A. Katrukha, and Lukas C. Kapitein
- Subjects
Polymerization ,Microtubule ,Chemistry ,Lattice defects ,Biophysics ,Nucleation ,Microtubule end ,Cytoskeleton ,Stabilizing Agents ,Microtubule severing - Abstract
Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.
- Published
- 2021
37. Mapping the neuronal cytoskeleton using expansion microscopy
- Author
-
Daphne, Jurriens, Vincent, van Batenburg, Eugene A, Katrukha, and Lukas C, Kapitein
- Subjects
Neurons ,Microscopy, Fluorescence ,Microtubules ,Cytoskeleton - Abstract
Expansion microscopy (ExM) is a recently introduced technique that enables high-resolution imaging with conventional microscopes by using physical expansion of samples. While this technique does not require a complicated microscope setup (like in STED or STORM microscopy), sample preparation and handling require additional attention. Here we describe a workflow for imaging of the neuronal microtubule network with minimal artifacts and sample perturbations. We demonstrate that the use of custom-printed mounting chambers simplifies sample handling and facilitates stable imaging of the sample. In addition, refractive index matching between the sample and the objective greatly improves signal retention deeper in thick samples. To accurately determine the precise expansion factor and determine sample distortion, we describe how samples can be compared using STED and ExM. Together, these procedures enabled us to better resolve different microtubule subsets in neuronal soma and dendrites.
- Published
- 2021
38. Mapping the neuronal cytoskeleton using expansion microscopy
- Author
-
Eugene A. Katrukha, Daphne Jurriens, Lukas C. Kapitein, and Vincent van Batenburg
- Subjects
0301 basic medicine ,Microscope ,Super-resolution microscopy ,STED microscopy ,Biology ,Signal ,Sample (graphics) ,law.invention ,03 medical and health sciences ,030104 developmental biology ,law ,Distortion ,Microscopy ,Biophysics ,Sample preparation ,Biological system - Abstract
Expansion microscopy (ExM) is a recently introduced technique that enables high-resolution imaging with conventional microscopes by using physical expansion of samples. While this technique does not require a complicated microscope setup (like in STED or STORM microscopy), sample preparation and handling require additional attention. Here we describe a workflow for imaging of the neuronal microtubule network with minimal artifacts and sample perturbations. We demonstrate that the use of custom-printed mounting chambers simplifies sample handling and facilitates stable imaging of the sample. In addition, refractive index matching between the sample and the objective greatly improves signal retention deeper in thick samples. To accurately determine the precise expansion factor and determine sample distortion, we describe how samples can be compared using STED and ExM. Together, these procedures enabled us to better resolve different microtubule subsets in neuronal soma and dendrites.
- Published
- 2021
39. Author response: Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells
- Author
-
Hugo G.J. Damstra, Wilhelmina E. van Riel, Florian Berger, Jorg van Loosdregt, Yesper Th Smits, Lukas C. Kapitein, Peter Jan Hooikaas, Maud Martin, Anna Akhmanova, and Oane J Gros
- Subjects
Physics ,Microtubule ,Centrosome ,Biophysics ,Kinesin ,Polarization (electrochemistry) - Published
- 2020
40. Author response: Concerted action of kinesins KIF5B and KIF13B promotes efficient secretory vesicle transport to microtubule plus ends
- Author
-
Ilya Grigoriev, Maud Martin, Ihor Smal, Qingyang Liu, Anna Akhmanova, Erik Meijering, Yao Yao, Andrea Serra-Marques, Lukas C. Kapitein, Veronika Solianova, Eugene A. Katrukha, Cathelijn A.E. Peeters, Peter Jan Hooikaas, and Lotte B. Pedersen
- Subjects
Action (philosophy) ,Microtubule ,Chemistry ,Kinesin ,Secretory Vesicle ,Cell biology - Published
- 2020
41. Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells
- Author
-
Hugo G.J. Damstra, Lukas C. Kapitein, Yesper Th Smits, Wilhelmina E. van Riel, Peter Jan Hooikaas, Anna Akhmanova, Jorg van Loosdregt, Florian Berger, Oane J Gros, and Maud Martin
- Subjects
medicine.anatomical_structure ,Microtubule ,Centrosome ,Chemistry ,T cell ,Organelle ,Cell ,medicine ,Kinesin ,Cytoskeleton ,Immunological synapse ,Cell biology - Abstract
When a T cell and an antigen-presenting cell form an immunological synapse, rapid dynein-driven translocation of the centrosome towards the contact site leads to reorganization of microtubules and associated organelles. Currently, little is known about how the regulation of microtubule dynamics contributes to this process. Here, we show that the knockout of KIF21B, a kinesin-4 linked to autoimmune disorders, causes microtubule overgrowth and perturbs centrosome translocation. KIF21B restricts microtubule length by inducing microtubule pausing typically followed by catastrophe. Catastrophe induction with vinblastine prevented microtubule overgrowth and was sufficient to rescue centrosome polarization in KIF21B-knockout cells. Biophysical simulations showed that a relatively small number of KIF21B molecules can restrict microtubule length and promote an imbalance of dynein-mediated pulling forces that allows the centrosome to translocate past the nucleus. We conclude that proper control of microtubule length is important for allowing rapid remodeling of the cytoskeleton and efficient T cell polarization.
- Published
- 2020
42. Co-translational targeting of transcripts to endosomes
- Author
-
Doris Popovic, Wilco Nijenhuis, Lukas C. Kapitein, and Lucas Pelkmans
- Abstract
Asymmetric localization and translation of mRNAs is used by single cells to sense their environment and integrate extrinsic cues with the appropriate cellular response. Here we investigate the extent to which endosomes impact subcellular patterning of transcripts and provide a platform for localized translation. Using image-based transcriptomics, indirect immunofluorescence, and RNAseq of isolated organelles, we discover mRNAs that associate with early endosomes in a translation-dependent and -independent manner. We explore this in more detail for the mRNA of a major endosomal tethering factor and fusogen, Early Endosomal Antigen 1, EEA1, which localizes to early endosomes in a puromycin-sensitive manner. By reconstituting EEA1 knock-out cells with either the coding sequence or 3’UTR of EEA1, we show that the coding region is sufficient for endosomal localization of mRNA. Finally, we use quantitative proteomics to discover proteins associated with EEA1 mRNA and identify CSRP1 as a factor that controls EEA1 translational efficiency. Our findings reveal that multiple transcripts associate with early endosomes in a translation-dependent manner and identify mRNA-binding proteins that may participate in controlling endosome-localized translation.
- Published
- 2020
43. REALM: AO-based localization microscopy deep in complex tissue
- Author
-
Marijn E. Siemons, Lukas C. Kapitein, Naomi A. K. Hanemaaijer, and Maarten H. P. Kole
- Subjects
Physics ,Microscopy ,Biophysics ,Brain tissue ,Cytoskeleton ,Adaptive optics ,Axon initial segment - Abstract
Performing Single-Molecule Localization Microscopy (SMLM) in complex biological tissues, where sample-induced aberrations hamper detection and localization, has remained a challenge. Here we establish REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of ≤1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. We demonstrate this method by resolving the periodic cytoskeleton of the axon initial segment at 50 μm depth in brain tissue.
- Published
- 2020
44. Concerted action of kinesins KIF5B and KIF13B promotes efficient secretory vesicle transport to microtubule plus ends
- Author
-
Lukas C. Kapitein, Eugene A. Katrukha, Peter Jan Hooikaas, Ihor Smal, Cathelijn A.E. Peeters, Qingyang Liu, Erik Meijering, Lotte B. Pedersen, Yao Yao, Andrea Serra-Marques, Maud Martin, Anna Akhmanova, Ilya Grigoriev, and Veronika Solianova
- Subjects
symbols.namesake ,Microtubule ,Chemistry ,Vesicle ,symbols ,Kinesin ,Secretion ,macromolecular substances ,Golgi apparatus ,Secretory Vesicle ,Intracellular transport ,Cell biology - Abstract
Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.
- Published
- 2020
45. The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes
- Author
-
David Holcman, Charles N. Christensen, Christine E. Holt, Lukas C. Kapitein, Pierre Parutto, Francesca W. van Tartwijk, Gabriele Kaminski Schierle, Edward Avezov, Julie Qiaojin Lin, Wilco Nijenhuis, Alan Tunnacliffe, Clemens F. Kaminski, Marcus Fantham, Meng Lu, Lu, Meng [0000-0001-9311-2666], van Tartwijk, Francesca [0000-0002-9795-2571], Lin, Qiaojin [0000-0002-2669-6478], Christensen, Charles [0000-0002-5355-1063], Avezov, Edward [0000-0002-2894-0585], Holt, Christine [0000-0003-2829-121X], Kaminski Schierle, Gabriele [0000-0002-1843-2202], Kaminski, Clemens [0000-0002-5194-0962], and Apollo - University of Cambridge Repository
- Subjects
Chemistry ,Endoplasmic reticulum ,Cell ,Metabolic change ,Nutritional status ,3101 Biochemistry and Cell Biology ,Cell biology ,medicine.anatomical_structure ,Tubule ,Global distribution ,Lysosome ,medicine ,Causal link ,health care economics and organizations ,31 Biological Sciences - Abstract
The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains, sheets and interconnected tubules. These domains undergo dynamic reshaping, in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling within minutes are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status. The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven re-positioning of lysosomes, which leads to both a redistribution of the ER tubules and its global morphology. Lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.
- Published
- 2020
46. Innentitelbild: Chemical Imaging of Hierarchical Porosity Formation within a Zeolite Crystal Visualized by Small‐Angle X‐Ray Scattering and In‐Situ Fluorescence Microscopy (Angew. Chem. 25/2021)
- Author
-
Matthias Filez, Martin Vesely, Ivan Garcia‐Torregrosa, Marianna Gambino, Özgün Attila, Florian Meirer, Eugene A. Katrukha, Maarten B. J. Roeffaers, Jan Garrevoet, Lukas C. Kapitein, and Bert M. Weckhuysen
- Subjects
General Medicine - Published
- 2021
47. Purification and Application of a Small Actin Probe for Single-Molecule Localization Microscopy
- Author
-
Roderick P, Tas, Trusanne G A A, Bos, and Lukas C, Kapitein
- Subjects
Microscopy, Fluorescence ,Actins ,Fluorescent Dyes - Abstract
The cytoskeleton is involved in many cellular processes. Over the last decade, super-resolution microscopy has become widely available to image cytoskeletal structures, such as microtubules and actin, with great detail. For example, Single-Molecule Localization Microscopy (SMLM) achieves resolutions of 5-50 nm through repetitive sparse labeling of samples, followed by Point-Spread-Function analysis of individual fluorophores. Whereas initially this approach depended on the controlled photoswitching of fluorophores targeted to the structure of interest, alternative techniques now depend on the transient binding of fluorescently labeled probes, such as the small polypeptide lifeAct that can transiently interact with polymerized actin. These techniques allow for simple multicolor imaging and are no longer limited by a fluorophore's blinking properties. Here we describe a detailed step-by-step protocol to purify, label, and utilize the lifeAct fragment for SMLM. This purification and labeling strategy can potentially be extended to a variety of protein fragments compatible with SMLM.
- Published
- 2017
48. Author response: Kinesin-4 KIF21B is a potent microtubule pausing factor
- Author
-
Lukas C. Kapitein, Eugene A. Katrukha, Sarah Bianchi, Wilhelmina E. van Riel, Anna Akhmanova, Albert J. R. Heck, Casper C. Hoogenraad, Ankit Rai, Qingyang Liu, and Michel O. Steinmetz
- Subjects
Microtubule ,Chemistry ,Kinesin ,Cell biology - Published
- 2017
49. Electroporation of COS-7 cells and functionalization of QDs v1
- Author
-
Eugene A Katrukha, not provided Marina Mikhaylova, not provided Hugo X van Brakel, not provided Paul M van Bergen en Henegouwen, not provided Anna Akhmanova, not provided Casper C Hoogenraad, and not provided Lukas C Kapitein
- Subjects
Chemistry ,Electroporation ,Biophysics ,Surface modification - Abstract
Detailed cell electroporation protocol from paper: Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dotsEugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein
- Published
- 2017
50. Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots
- Author
-
Anna Akhmanova, Lukas C. Kapitein, Hugo X van Brakel, Eugene A. Katrukha, Paul M.P. van Bergen en Henegouwen, Marina Mikhaylova, Casper C. Hoogenraad, Sub Cell Biology, and Celbiologie
- Subjects
0301 basic medicine ,Cytoplasm ,Science ,General Physics and Astronomy ,Biological Transport, Active ,Kinesins ,macromolecular substances ,Myosins ,Biology ,010402 general chemistry ,01 natural sciences ,Microtubules ,General Biochemistry, Genetics and Molecular Biology ,Article ,Motor protein ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Chlorocebus aethiops ,Quantum Dots ,Animals ,Cytoskeleton ,Actin ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Chemistry ,technology, industry, and agriculture ,Biological Transport ,General Chemistry ,equipment and supplies ,Fick's laws of diffusion ,Actins ,0104 chemical sciences ,3. Good health ,Cell biology ,Actin Cytoskeleton ,030104 developmental biology ,Quantum dot ,COS Cells ,Biophysics ,Kinesin ,030217 neurology & neurosurgery ,Intracellular - Abstract
The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics., Methods to probe transport dynamics within cells can shed insight into the nature of the cytoplasm. Here the authors develop a method to functionalize and deliver quantum dots intracellularly to show how the cytoskeleton influences non-equilibrium intracellular transport dynamics.
- Published
- 2016
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