Biology subjects

Kaech, A.

Publications and source records attributed to Kaech, A..

8 recordsLinked to original sources

bioRxiv2026

Automated LN2 refill device for uninterrupted cryoFIB-SEM operations.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

biophysics↗

bioRxiv2024

Astrocytes adopt a progenitor-like migratory strategy for regeneration in adult brain

Mature astrocytes become activated upon non-specific tissue damage and contribute to glial scar formation. Proliferation and migration of adult reactive astrocytes after injury is considered very limited. However, the regenerative behavior of individual astrocytes following selective astroglial loss, as seen in astrocytopathies, such as neuromyelitis optica spectrum disorder, remains unexplored. Here, we performed longitudinal in vivo imaging of cortical astrocytes after focal astrocyte ablation in mice. We discovered that perilesional astrocytes develop a remarkable plasticity for efficient lesion repopulation. A subset of mature astrocytes transforms into reactive progenitor-like (REPL) astrocytes that not only undergo multiple asymmetric divisions but also remain in a multinucleated interstage. This regenerative response facilitates efficient migration of newly formed daughter cell nuclei towards unoccupied astrocyte territories. Our findings define the cellular principles of astrocyte plasticity upon focal lesion, unravelling the REPL phenotype as a fundamental regenerative strategy of mature astrocytes to restore astrocytic networks in the adult mammalian brain. Promoting this regenerative phenotype bears therapeutic potential for neurological conditions involving glial dysfunction.

neuroscience↗

bioRxiv2024

Coordination between ESCRT function and Rab conversion during endosome maturation

The endosomal pathway is essential for regulating cell signaling and cellular homeostasis. Rab5-positive early endosomes receive proteins from the plasma membrane. Dependent on a ubiquitin mark on the protein, they will be either recycled or sorted into intraluminal vesicles (ILVs) by ESCRT. During endosome maturation Rab5 will be replaced by Rab7 on endosomes that are able to fuse with lysosomes to form endolysosomes. Whether ESCRT-driven ILV formation and Rab5-to-Rab7 conversion are coordinated remains unknown. Here we show that loss of early ESCRTs leads to enlarged Rab5-positive endosomes and prohibit Rab conversion. Reduction of ubiquitinated cargo alleviated this phenotype. Moreover, ubiquitinated proteins on the endosomal limiting membrane prevented the displacement of the Rab5GEF RABX-5 by the Rab7GEF SAND-1/CCZ-1. Overexpression of Rab7 could partially overcome this block, even in the absence of SAND-1, whereby the HOPS complex presumably acts as Rab7GEF. Our data reveal a hierarchy of events in which cargo corralling by ESCRTs is upstream of Rab conversion and thereby ESCRT-0 and ubiquitinated cargo could act as timers determining the onset of Rab conversion.

cell biology↗

bioRxiv2021

Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria (MMAuria), present unique challenges to energetic homeostasis by disrupting energy producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut) type MMAuria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared to littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these indicate hypometabolism, energetic inflexibility and increased stores at the expense of active tissue as energy shortage consequences.

physiology↗

bioRxiv2021

The C. elegans PTCHD homolog PTR-4 is required for proper organization of the pre-cuticular apical extracellular matrix

The Patched-related (PTR) superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other PTR or Patched domain (PTCHD) family members. C. elegans has a large number of PTR/PTCHD proteins, despite lacking a canonical hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the pre-cuticle apical extracellular matrix (aECM), a transient and molecularly distinct aECM that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with pre-cuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when pre-cuticle matrix is present. Finally, PTR-4 acts in a cell non-autonomous manner to properly localize the secreted ZP domain protein LET-653 to the pre-cuticle aECM. We propose that PTR-4 exports lipids or other hydrophobic components of the pre-cuticle aECM.

cell biology↗

bioRxiv2020

Independent apical and basal mechanical systems determine cell and tissue shape in the Drosophila wing disc

How cell shape and mechanics are organized in three dimensions during tissue morphogenesis is poorly understood. In the Drosophila wing imaginal disc, we examined the mechanical processes that determine the shape of epithelial cells. Since it has been known that basement membrane influences the mechanics intracellularly, we reexamined the material properties of the basement membrane with fluorescence and transmission electron microscopy in its native environment. Further, we investigated the effect on cell shape and tissue mechanics when disruptions were instigated at three different time scales: (1) short (seconds with laser cutting), (2) medium (minutes with drug treatments), and (3) long (days with RNAi interference). We found regions in which the basement membrane is much thicker and heterogeneous than previously reported. Disrupting the actin cytoskeleton through drug treatment affects cell shape only at the apical surface, while the shapes in the medial and basal surfaces were not altered. In contrast, when integrin function was inhibited via RNAi or basement membrane integrity was disrupted by drug treatment, the medial and basal cell shapes were affected. We propose that basement membrane thickness patterns determine the height and basal surface area of cells and the curvature of folds in the wing disc. Based on these findings and previous studies, we propose a model of how cell shapes and tissue properties were determined by highly local, modular apical and basal mechanics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/036152v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@177c8f0org.highwire.dtl.DTLVardef@1292624org.highwire.dtl.DTLVardef@871577org.highwire.dtl.DTLVardef@925bcd_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Cytoplasmic flows caused by actomyosin contraction drive interkinetic nuclear migration

Interkinetic nuclear migration (IKNM) is the process by which the nucleus migrates between apical and medial surfaces of pseudostratified epithelia. Previous studies have proposed force generating mechanisms, acting primarily on the nucleus. Having observed in drosophila wing discs that cytoplasmic components (lipid droplets and mitochondria) migrate alongside the nucleus, we used live imaging and particle tracking to demonstrate that the cytoplasm flows are responsible for the nucleus migration. We identify that nuclear migration in mitotic cells is preceded by a fast basal-to-apical flow of cytoplasm occurring over short time scales. We further show that, for the migration of basally located nuclei to an apical position, a slower flow of cytoplasm is responsible over a longer time scale. Our findings indicate that these flows are driven by acto-myosin contractile forces. These flows increase the hydrostatic pressure under the nucleus to exert a lifting force, much like a piston in a hydraulic cylinder.

biophysics↗

bioRxiv2019

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

AbstractHedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat storage and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPAR homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a novel role for PTCH in organelle structure maintenance and fat metabolism.

cell biology↗