Biology subjects

Joubert, L.-M.

Publications and source records attributed to Joubert, L.-M..

8 recordsLinked to original sources

bioRxiv2025

Cryoprotectants-assisted plunge freezing of thick brain tissue specimens for targeted physiologically relevant cryo-imaging in situ

In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. As a result, cryo-imaging reports obtained from non-trivial specimens including mammalian brain tissues are scarce and their physiological relevance remains to be determined. Here, we benchmarked plunge freezing with a variety of cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 microns thick and across several brain regions while keeping the tissue functional. By utilizing the knock-in (KI) mouse model with fluorescent astrocytes we have performed targeted cryo-FIB/SEM volume-EM imaging as well as targeted high-resolution cryoET imaging. Prior to cryoET, we have successfully generated lamellae in a semi-automated fashion on both LMIS (liquid metal ion source)- and plasma-based cryo-FIB/SEM instrumentation thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and astrocytes processes and validated the physiological relevance of our outputs based on the morphology of the corresponding cellular and subcellular features. The pipeline utilizes common vitrification setups and can be potentially extended toward alternative tissue specimens. Ultimately, we expect our approach to become an important step towards democratization of physiologically relevant in situ cryo-imaging studies.

biophysics↗

bioRxiv2024

Illuminating microbial mat assembly: Cyanobacteria and Chloroflexota cooperate to structure light-responsive biofilms

Microbial mats are stratified communities often dominated by unicellular and filamentous phototrophs within an exopolymer matrix. It is challenging to quantify the dynamic responses of community members in situ as they experience steep gradients and rapid fluctuations of light. To address this, we developed a binary consortium using two representative isolates from hot spring mats: the unicellular oxygenic phototrophic cyanobacterium Synechococcus OS-B (Syn OS-B) and the filamentous anoxygenic phototroph Chloroflexus MS-CIW-1 (Chfl MS-1). We quantified the motility of individual cells and entire colonies and demonstrated that Chfl MS-1 formed bundles of filaments that moved in all directions with no directional bias to light. Syn OS- B was slightly less motile but exhibited positive phototaxis. This binary consortium displayed cooperative behavior by moving further than either species alone and formed ordered arrays where both species aligned with the light source. No cooperative motility was observed when a non-motile pilB mutant of Syn OS-B was used instead of Syn OS-B. The binary consortium also produced more adherent biofilm than individual species, consistent with the close interspecies association revealed by electron microscopy. We propose that cyanobacteria and Chloroflexota cooperate in forming natural microbial mats, by colonizing new niches and building robust biofilms. SignificanceMicrobial mats are dense, layered communities with ancient origins and widespread occurrence, but how they assemble is not well understood. To investigate how microbial motility, physical interactions, and responses to light affect mat assembly, we developed a binary consortium from representative hot spring mat isolates. Individually, the Cyanobacteria and Chloroflexota isolates displayed significant differences in motility and biofilm formation. When combined, the consortium exhibited enhanced motility towards light and formed more robust biofilms. This model consortium approach complements in situ studies by directly testing the role of motility and physical cooperation in shaping microbial mats, and could inform biofilm applications in industrial settings.

microbiology↗

bioRxiv2024

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for [~]50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cells apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cells affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

plant biology↗

bioRxiv2024

Structural characterization and dynamics of AdhE ultrastructures from Clostridium thermocellum: A containment strategy for toxic intermediates?

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe, however ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 [A] structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

biophysics↗

bioRxiv2023

Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts

Parabasalid protists recently emerged as keystone members of the mammalian microbiota with important effects on their hosts health. However, the prevalence and diversity of parabasalids in wild reptiles and the consequences of captivity and other environmental factors on these symbiotic protists are unknown. Reptiles are ectothermic, and their microbiomes are subject to temperature fluctuations, such as those driven by climate change. Thus, conservation efforts for threatened reptile species may benefit from understanding how shifts in temperature and captive breeding influence the microbiota, including parabasalids, to impact host fitness and disease susceptibility. Here, we surveyed intestinal parabasalids in a cohort of wild reptiles across three continents and compared these to captive animals. Reptiles harbor surprisingly few species of parabasalids compared to mammals, but these protists exhibited a flexible host-range, suggesting specific adaptations to reptilian social structures and microbiota transmission. Furthermore, reptile-associated parabasalids are adapted to wide temperature ranges, although colder temperatures significantly altered the protist transcriptomes, with increased expression of genes associated with detrimental interactions with the host. Our findings establish that parabasalids are widely distributed in the microbiota of wild and captive reptiles and highlight how these protists respond to temperature swings encountered in their ectothermic hosts.

microbiology↗

bioRxiv2022

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

Huntingtons Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics to show differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

neuroscience↗

bioRxiv2017

Wide-field dynamic monitoring of immune cell trafficking in murine models of glioblastoma

Leukocyte populations, especially tumor-associated macrophages (TAMs), are capable of mediating both anti- and pro-tumor processes and play significant roles in the tumor microenvironment. Moreover, TAMs have been shown to exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. Laboratory investigation into the behavior of TAMs has been limited by a lack of methods capable of resolving the in vivo distribution and dynamics of this cell population across wide fields of view. Recent studies have employed magnetic resonance imaging and intravital microscopy in conjunction with nanoparticle labeling methods to detect TAMs and observe their responses to therapeutic agents. Here we describe a novel method to enable high-resolution, wide-field, longitudinal imaging of leukocytes based on contrast-enhanced Speckle-Modulating Optical Coherence Tomography (SM-OCT), which substantially reduces imaging noise. We were able to specifically label TAMs and activated microglia in vivo with large gold nanorod contrast agents (LGNRs) in an orthotopic murine glioblastoma model. After labeling, we demonstrated near real-time tracking of leukocyte migration and distribution within the tumors. The intrinsic resolution, imaging depth, and sensitivity of this method may facilitate detailed studies of the fundamental behaviors of TAMs in vivo, including their intratumoral distribution heterogeneity and the roles they play in modulating cancer proliferation. In future studies, the method described herein may also provide the necessary means to characterize TAM responses to immunotherapeutic regimens in a range of solid tumors.

bioengineering↗

bioRxiv2017

Revealing The Cell-Material Interface With Nanometer Resolution By FIB-SEM

The interface between biological cells and non-biological surfaces profoundly influences cellular activities, chronic tissue responses, and ultimately the success of medical implants. Materials in contact with cells can be plastics, metal, ceramics or other synthetic materials, and their surfaces vary widely in chemical compositions, stiffness, topography and levels of roughness. To understand the molecular mechanism of how cells and tissues respond to different materials, it is of critical importance to directly visualize the cell-material interface at the relevant length scale of nanometers. Conventional ultrastructural analysis by transmission electron microscopy (TEM) often requires substrate removal before microtome sectioning, which is not only challenging for most substrates but also can cause structural distortions of the interface. Here, we present a new method for in situ examination of the cell-to-material interface at any desired cellular location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM). This method involves a thin-layer plastification procedure that preserves adherent cells as well as enhances the contrast of biological specimen. We demonstrate that this unique procedure allows the visualization of cell-to-material interface and intracellular structures with 10nm resolution, compatible with a variety of materials and surface topographies, and capable of volume and multi-directional imaging. We expect that this method will be very useful for studies of cell-to-material interactions and also suitable for in vivo studies such as examining osteoblast adhesion and new bone formation in response to titanium implants.

biophysics↗