Biology subjects

Nievergelt, A. P.

Publications and source records attributed to Nievergelt, A. P..

8 recordsLinked to original sources

bioRxiv2024

Unwrapping the ciliary coat: high-resolution structure and function of the ciliary glycocalyx

The glycocalyx, a highly heterogeneous glycoprotein layer of cilia regulates adhesion and force transduction, and is involved in signalling. The high-resolution molecular architecture of this layer is currently not understood. We describe the structure of the ciliary coat in the green alga Chlamydomonas reinhardtii by cryo-electron tomography and proteomic approaches and present the high-resolution cryoEM structure of the main component, FMG1B. We describe FMG1B as a mucin orthologue which lacks the major O-glycosylation of mammalian mucins, but is N-glycosylated. FMG1A, a previously undescribed isoform of FMG1B is expressed in C. reinhardtii. By microflow-based adhesion assays we observe increased surface adhesion in the glycocalyx deficient double-mutant fmg1a-fmg1b. We find this mutant to be capable of surface-gliding, with neither isoform required for extracellular force transduction by intraflagellar transport. Our results find FMG1 to form a protective layer with adhesion-regulative instead of adhesion-conferring properties and an example of an undescribed class of mucins.

cell biology↗

bioRxiv2024

Tubulin tyrosination/detyrosination governs the spatial segregation of intraflagellar transport trains on axonemal microtubule doublets

Assembly and function of cilia rely on the continuous transport of ciliary components between the cell body and the ciliary tip. This is performed by specialized molecular machines, known as Intraflagellar Transport (IFT) trains. Anterograde IFT trains are powered by kinesin-2 motors and move along the B-tubules (enriched in detyrosinated tubulin) of the ciliary microtubule doublets. Conversely, retrograde IFT trains are moved by dynein-1b motors along the A-tubules (enriched in tyrosinated tubulin) back to the cell body. The segregation of oppositely directed trains on A-tubules or B-tubules is thought to prevent traffic jams in the cilium, but the mechanism by which opposite polarity trains are sorted onto either tubule, and whether tubulin tyrosination/detyrosination plays a role in that process, is unknown. Here, we show that CRISPR-mediated knock-out of VashL, the enzyme that detyrosinates microtubules, causes recurrent stoppages of IFT trains and reduces the rate of ciliary growth in Chlamydomonas reinhardtii. To test whether the observed stoppages, potentially caused by collisions between oppositely directed IFT trains, are ascribable to direct interactions between IFT trains and tubulin tyrosination/detyrosination, we developed methods to reconstitute the motility of native IFT trains from cilia on de-membranated axonemes and ex vivo on synthetically polymerized microtubules. We show that anterograde trains have higher affinity for detyrosinated microtubules (analogous to B-tubules), while retrograde trains for tyrosinated microtubules (analogous to A-tubules). We conclude that tubulin tyrosination/detyrosination is required for the spatial segregation of oppositely directed trains and for their smooth uninterrupted motion. Our results provide a model for how the tubulin code governs molecular transport in cilia.

cell biology↗

bioRxiv2023

The Tubulin Nano-Code: a protofilament-specific pattern of tubulin post-translational modifications regulates ciliary beating mechanics

Control of ciliary beating is crucial for motility and signaling in eukaryotic cells and requires spatially restricted interactions between axonemal proteins and specific protofilaments within the ciliary microtubules. How these interactions are regulated remains poorly understood, but increasing evidence indicates that tubulin post-translational modifications (tPTMs) are required for proper ciliary motility. The Tubulin Code refers to the concept that tPTMs can modulate the function of individual microtubules in cells. Here we use a combination of immuno-cryo-electron tomography, expansion microscopy and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nano-patterns at sub-microtubular scale. We show that these two nano-patterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are required for their regulation during ciliary beating. Our discovery of a tubulin nano-code in cilia highlights the need of higher-resolution studies also in other cellular compartments to understand the molecular role of tPTMs. One-Sentence SummaryTubulin post-translational modifications form nanopatterns at sub-microtubular scale that enable individual protofilaments to perform specific functions Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/546853v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@253704org.highwire.dtl.DTLVardef@13f0a8forg.highwire.dtl.DTLVardef@1ce673dorg.highwire.dtl.DTLVardef@1dc04e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Efficient precision genome editing of Chlamydomonas reinhardtii with CRISPR/Cas

CRISPR/Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now only been applied to targeted gene disruption, whereas scar-less knock-in transgenesis has generally been considered infeasible. We have developed highly efficient homology-directed knock-in mutagenesis in cell-walled strains of Chlamydomonas. Our method allows scarless integration of fusion tags and sequence modifications of near arbitrary proteins without need for a preceding mutant line.

cell biology↗

bioRxiv2021

Time-resolved scanning ion conductance microscopy for three-dimensional tracking of nanoscale cell surface dynamics

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy1, EM2, AFM3). However, time-resolved imaging techniques tend to either have lower resolution, are limited to small areas, or cause damage to the cells thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging. By utilizing recent advances in nanopositioning4, nanopore fabrication5, microelectronics6, and controls engineering7 we developed a microscopy method that can resolve spatiotemporally diverse three-dimenional processes on the cell membrane at sub-5nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of sub-second to days, imagining diverse processes ranging from endocytosis, micropinocytosis, and mitosis, to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

biophysics↗

bioRxiv2021

Intraflagellar transport trains can turn around without the ciliary tip complex

Cilia and flagella are microtubule doublet based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavourable to diffusive transport for their assembly and maintenance. Instead, a highly dynamic system of intraflagellar transport (IFT) trains moves rapidly up and down the cilium. However, the mechanism of how these trains turn around upon reaching the ciliary tip has remained elusive. It has been hypothesized that there exists a dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate this conversion. In this work, we use physical and chemical methods to manipulate IFT in the cilia of the unicellular green alga Chlamydomonas reinhardtii to show that no such stationary tip-machinery is required for IFT turnaround. Instead, we demonstrate that the conversion from anterograde to retrograde IFT trains is a calcium independent intrinsic ability of the IFT system.

cell biology↗

bioRxiv2020

Surface-catalyzed SAS-6 self-assembly directs centriole formation through kinetic and structural mechanisms

Discovering the physical principles directing organelle assembly is a fundamental pursuit in biology. Centrioles are evolutionarily conserved organelles with a 9-fold rotational symmetry of chiral microtubules imparted onto the cilia they template1. Centriole assemble from likewise symmetrical ring polymers of SAS-6 proteins, orthogonal to a toroidal surface surrounding the resident centriole2-4. How surface properties ensure ring assembly with proper symmetry and orthogonal arrangement is not known. Here, we deployed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to decipher physical principles of surface-guided SAS-6 self-assembly. Using machine learning to quantify the polymerization reaction and developing a coagulation-fragmentation model, we discovered that the surface shifts the reaction equilibrium by [~]104 compared to the solution situation, explaining orthogonal organelle emergence. Moreover, molecular dynamics and PORT-HS-AFM revealed that the surface converts helical SAS-6 polymers into 9-fold ring polymers with residual asymmetry, which may impart chiral features to centrioles and cilia. Overall, we discovered two fundamental physical principles directing robust centriole organelle assembly.

biophysics↗

bioRxiv2018

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b6 interacts with the lipid bilayer prior to recruiting C7 and C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

biophysics↗