Biology subjects

Kapitein, L. C.

Publications and source records attributed to Kapitein, L. C..

30 records · Page 2Linked to original sources

bioRxiv2021

Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

cell biology↗

bioRxiv2021

Centriole-independent centrosome assembly in interphase mammalian cells

AbstractThe major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein{-}mediated PCM transport, but the self-assembly properties of PCM in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based PCM transport and PCM self-clustering are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase PCM self-assembly depends on {gamma}-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152 or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor-and microtubule-based rearrangement.

cell biology↗

bioRxiv2021

CFTR modulator response measurements in subjects with cystic fibrosis using 2D differentiated nasal epithelia converted into spheroids

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-1{beta} and interleukine-1{beta}, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF.

cell biology↗

bioRxiv2021

A live-cell marker to visualize the dynamics of stable microtubules

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.

cell biology↗

bioRxiv2021

Microtubule lattice defects promote catastrophes.

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.

cell biology↗

bioRxiv2021

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

cell biology↗

bioRxiv2020

Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells.

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.

cell biology↗

bioRxiv2020

REALM: AO-based localization microscopy deep in complex tissue

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 m depth in brain tissue.

cell biology↗

bioRxiv2020

Concerted action of kinesin-1 KIF5B and kinesin-3 KIF13B promotes efficient transport of exocytotic vesicles to microtubule plus ends

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.

cell biology↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗

bioRxiv2018

MAP7 family proteins are microtubule-tethered allosteric activators of kinesin-1.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1 and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared to MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be co-transported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.\n\nSummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.

cell biology↗

bioRxiv2016

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization 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 transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle directional fluctuations, because the specific subset of stable microtubules used by this motor underwent prominent undulations. Using actin-targeting agents we found that the actin network suppresses most microtubule shape remodeling, rather than promoting them. These results demonstrate how the spatial and structural heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

biophysics↗