Biology subjects

Kea-te Lindert, M.

Publications and source records attributed to Kea-te Lindert, M..

6 recordsLinked to original sources

bioRxiv2024

Development of an Organ-on-a-Chip for Correlative Microscopy: Visualizing Early Osteogenesis in 3D with High Resolution

Correlative microscopy approaches offer powerful means to study tissue development across spatial scales, but combining 3D light and electron imaging remains technically challenging. Here, we present a practical workflow that integrates organ-on-a-chip culture with longitudinal fluorescence imaging and volume electron microscopy. By modifying an existing chip platform designed for aligned tissue growth, we demonstrate the feasibility of extended 3D live imaging and subsequent high-pressure freezing of intact microtissues. Fluorescence-guided targeting enables focused ion beam/scanning electron microscopy (FIB/SEM) of selected regions, revealing ultrastructural features such as cellular organization, collagen alignment, and matrix mineralization. While not aimed at new biological discoveries, this study highlights the compatibility and potential of this pipeline for future high-resolution, multiscale studies of tissue morphogenesis and pathology in controlled microenvironments.

bioengineering↗

bioRxiv2023

Osteoblast-induced collagen alignment in a 3D in vitro bone model

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized by hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we developed a 3D cell culture system in which osteogenic cells deposit an oriented collagen matrix, that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenic cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration towards the periphery of the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenesis and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

cell biology↗

bioRxiv2023

Comparative 3D ultrastructure of Plasmodium falciparum gametocytes

Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. In this study, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure of Plasmodium falciparum gametocytes of both genders and in different stages of development as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome variant. Furthermore, we generate, among other organelles, the first 3D reconstructions of endoplasmic reticulum (ER), Golgi apparatus, and extraparasitic structures in gametocytes. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar membrane of the gametocyte. Public availability of these volumetric electron microscopy resources of wild-type asexual and sexual blood-stage malaria parasites will facilitate reinterrogation of this global dataset with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure of P. falciparum gametocytes in high detail and shed light on the unique organellar biology of these deadly parasites.

microbiology↗

bioRxiv2021

Catestatin regulates the colonic mucus layer in inflammatory bowel disease

BackgroundThe pro-hormone chromogranin A (CgA) and its bioactive cleavage product catestatin (CST) are both associated with inflammatory bowel disease (IBD) and dysregulated barrier functions, but their exact role has remained elusive. Here, we demonstrate that CST regulates the colonic mucus layer. MethodsCST levels were measured in feces of IBD patients. The mucus layer, goblet cells, and immune cell infiltration were analyzed by histology and electron microscopy in colon tissue from IBD patients and mice with selective deletion of the CST-coding region of the CgA gene. ResultsCST levels were elevated in feces of IBD patients compared to healthy controls. The thickness of the mucus layer was increased in non-affected, but not in inflamed, regions of the colon in IBD patients. The thickness of the mucus layer and concomitant mucus production were also increased in the CST-KO mouse. This mucus phenotype in CST-KO mice could be reversed by bone marrow transplantation from wildtype mice. ConclusionsCST produced by bone-marrow derived immune cells reduces production of the mucus layer in the intestine. This might contribute to the reduced mucus layer in inflamed colon regions of IBD patients. Additionally, CST feces levels might be a biomarker for IBD.

immunology↗

bioRxiv2020

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

microbiology↗

bioRxiv2020

HPM Live μ for full CLEM workflow

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

biophysics↗