Biology subjects

Reinhardt, D. P.

Publications and source records attributed to Reinhardt, D. P..

9 recordsLinked to original sources

bioRxiv2026

Structural tuning of native type V fimbriae shapes mechanical specialization in Porphyromonas gingivalis

Fimbriae are proteinaceous filaments central to bacterial adhesion, colonization, and biofilm formation. Porphyromonas gingivalis, a keystone periodontal pathogen, utilizes two type V fimbriae systems, the major Fim and the minor Mfa fimbriae, that support distinct adhesive and biofilm-associated functions. Here, we determine the high-resolution cryo-electron microscopy structures of natively assembled FimA and Mfa1 stalks purified directly from P. gingivalis. Despite a shared donor-strand exchange mechanism, the two stalks exhibit distinct helical geometries and donor-strand environments. FimA has a larger helical pitch, whereas the Mfa1 donor strand is more extensively buried and further shielded by an ordered N-terminal latch. Comparison with monomeric stalk pilin structures reveals distinct assembly-associated remodeling around the donor-strand interface. Deletion of the Mfa1 latch reduces heat-resistant oligomer accumulation and produces species consistent with incomplete processing, yet recovered filaments retain the donor-strand-exchanged stalk architecture and exhibit initial force peaks comparable to native Mfa1 during simulated axial extension. Atomic force microscopy reveals greater apparent local stiffness of FimA under indentation, whereas steered molecular dynamics simulations show multiple lower-force axial transitions in FimA but a dominant higher-force transition in Mfa1. In both systems, the exchanged donor strands remain engaged during simulated extension while distinct surrounding contact networks rearrange. Together, these findings demonstrate how a conserved polymerization linkage can support structurally and mechanically distinct adhesive filaments, providing a molecular basis for mechanical diversification of type V fimbriae.

molecular biology↗

bioRxiv2025

Muscle stem cells produce a protective Fibrillin-1 matrix to prevent precocious activation.

Multiple biological mechanisms have been uncovered to regulate muscle stem cell quiescence, including inhibition of differentiation, adhesion-dependent anchoring, and translational control, which can be broadly classified as intrinsic or extrinsic properties. Here, we identify the matrix glycoprotein Fibrillin-1 (FBN1) as a Notch-regulated, cell-autonomous effector, essential for maintaining quiescence in muscle stem cells. Known for its causal role in Marfan syndrome (MFS), a connective tissue disorder that also presents with skeletal muscle atrophy, our work positions FBN1 as a critical niche component that protects stem cells from aberrant growth factor signalling. We demonstrate that targeted deletion of Fbn1 in muscle stem cells leads to dose-dependent quiescence defects, characterized by loss of cellular projections, depletion of the stem cell pool, and progressive decline in muscle function. Consistently, human MFS muscle biopsies show abnormally activated satellite cells, implicating stem cell imbalance in the development of MFS-associated myopathy. Mechanistically, the loss of FBN1 upregulates TGF{beta} signalling, and pharmacological inhibition of this pathway using the Angiotensin Receptor blocker losartan restores the cellular and physiological defects of mutant muscles. These findings reveal a new quiescence-preserving mechanism through extracellular matrix-mediated shielding from mitogenic signals, and position stem cell dysfunction as a driver of MFS myopathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/666604v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1895e71org.highwire.dtl.DTLVardef@1974e87org.highwire.dtl.DTLVardef@a49979org.highwire.dtl.DTLVardef@5813c9_HPS_FORMAT_FIGEXP M_FIG C_FIG Model of Fibrillin-1 protective barrier in quiescent satellite cells.Satellite cells produce Fibrillin-1 (FBN1) to establish a localized extracellular matrix barrier that limits exposure to mitogenic signals. Mechanistically, FBN1 expression is induced by Notch signalling and serves to sequester latent TGF{beta} complexes in an inactive form, thereby preventing their activation within the immediate niche. This barrier is spatially restricted beneath the basal lamina and remains functionally independent from the abundant interstitial FBN1 produced by fibro-adipogenic progenitors (FAPs). The model describes a self-contained, satellite cell-derived ECM system that maintains quiescence by insulating the cells from activating cues.

cell biology↗

bioRxiv2024

Stored Elastic Bending Tension as a Mediator of Embryonic Body Folding

During development, amniote vertebrate embryos transform from a flat, multi-layered sheet into a three-dimensional cylindrical form, through ventral folding of the lateral sides of the sheet (the lateral plate, LP) and their fusion in the ventral midline. Although this basic aspect of vertebrate body plan formation has long been described, it is not understood at the mechanistic level. Each side of the LP is comprised of two tissue layers: a dorsal somatopleure (Sop) of pseudostratified coelomic epithelium, extracellular matrix (ECM) and ectoderm, and a ventral splanchnopleure (Spl) of similar construction except with endoderm instead of ectoderm. Using a chick embryo slice system we find that the flat stage is actually a poised balance of opposing elastic bending tensions, with dorsal bending tension in the Sop opposing ventral bending tension in the Spl. An intact extracellular matrix is required for generating the bending tensions, as localized enzymatic digestion of Sop or Spl ECM dissipates tension, while removal of the endodermal or ectodermal layers has no effect. As development proceeds, the Sop undergoes Epithelial-Mesenchymal Transition, ECM fragmentation and dissipation of dorsal bending tension, while the Spl ECM and ventral bending tension remain intact, thus changing the balance of bending forces in the LP to promote ventral folding. Consistent with these findings, interference with the elastic ECM component fibrillin in the Spl in vivo reduces stored bending tension and perturbs ventral body folding. A generalizable conceptual model is presented in which embryonic growth, in the context of specific embryonic geometrical constraints, leads to accumulation of bending tension in the LP ECM, which is harnessed to drive body folding.

developmental biology↗

bioRxiv2024

Role of Fibronectin in Postnatal Skeletal Development

Fibronectin (FN) is a ubiquitous matrix glycoprotein essential for the physiological development of various tissues. Mutations in FN cause a form of skeletal dysplasia, emphasizing the importance of FN in cartilage and bone development. However, the relevance and functional role of FN during skeletal development remains elusive. We employed conditional knockout mouse models for the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and a double knockout (FNdKO) to determine the relevance of these two principal FN isoforms in postnatal skeletal development spanning from P1 to 2 months of age. We identified a unique topological FN deposition pattern in the mouse limb with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Circulating pFN did not enter the growth plate and was limited to the primary ossification center, whereas cartilage-specific cFN was detected as the major isoform in epiphyseal cartilage. Deletion of either one of the isoforms in single knockouts (cFNKO or pFNKO) only led to subtle changes in some of the analyzed parameters. Complete ablation of both cFN in the growth plate and circulating pFN in plasma resulted in significantly reduced postnatal body weight, body length, and bone length in the FNdKO mice. Assessment of the FNdKO adult bone microarchitecture using micro-CT revealed significantly reduced trabecular bone volume, trabecular network, bone mineral density, and increased bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice showed changes in the proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and type X, reduced apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. FNdKO mice also displayed decreased levels of transforming growth factor-{beta}1 (TGF{beta}1) and downstream phospho-AKT levels, which are critical regulators of chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for proper chondrogenesis and postnatal bone development. Simultaneous deletion of both FN isoforms in the developing cartilage leads to critical TGF{beta}-mediated alterations in chondrogenic differentiation, resulting in bone and skeletal defects. Significance/HighlightsO_LIFN is highly expressed during mouse limb development with increased deposition in resting and hypertrophic chondrocyte zones and the primary ossification center. C_LIO_LICartilage-specific cFN and circulating pFN are distinctly distributed during embryonic and postnatal bone development, with chondrocyte-specific cFN present in the growth plate and pFN limited to trabecular bone and the bone marrow. C_LIO_LIDeletion of both cFN and pFN leads to reduced bone growth during early postnatal development. C_LIO_LIDeletion of cFN and pFN leads to reduced trabecular bone formation, bone mineralization, and increased bone marrow adiposity in 2-month adult mice. C_LIO_LIAbsence of both FN isoforms in the FNdKO mouse model leads to altered chondrogenesis and reduced bone formation. C_LIO_LIFN regulates chondrogenesis via TGF{beta}-mediated phospho-AKT signaling. C_LI

developmental biology↗

bioRxiv2023

Mutations in Fibronectin Dysregulate Chondrogenesis in Corner Fracture Type Spondylometaphyseal Dysplasia

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER and transitioned into lysosomes. Massive amounts of these vesicles were not cleared from the cytosol. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. FN mutant cells also displayed altered FN splice variants under chondrogenic stimuli. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGF{beta}1) expression, crucial for mesenchymal condensation. Exogenous FN or TGF{beta}1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. Significance /Highlights* SMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells. * Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, which transition into lysosomes. * The cells cannot clear the massive accumulation of cytosolic vesicles. * Mutations in fibronectin impair stem cell proliferation, mesenchymal condensation, and the differentiation of MSCs into chondrocytes. * Exogenous addition of purified fibronectin or TGF{beta}-1 improves mesenchymal condensation and chondrogenesis of the FN mutant stem cells.

cell biology↗

bioRxiv2023

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz Syndrome patients provides insights into genotype-phenotype relations

IntroductionPathogenic (P) and likely pathogenic (LP) variants in the SMAD3 gene cause Loeys-Dietz syndrome type 3 (LDS3), also known as aneurysms-osteoarthritis syndrome (AOS). The phenotype of LDS3 is highly variable and characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with skeletal, cutaneous and facial features. ObjectivesInvestigate the impact of P/LP SMAD3 variants through conducting functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs).The resulting knowledge will optimize interpretation of SMAD3 variants. Material and methodsWe conducted a retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and utilized patient-derived VSMCs to investigate the functional impacts of dominant negative (DN) and haploinsufficient (HI) variants in SMAD3. Additionally, to broaden our cell model accessibility, we performed similar functional analyses on patient-derived fibroblasts carrying SMAD3 variants, differentiating them into myofibroblasts with the same variants. This enabled us to study the functional effects of DN and HI variants in SMAD3 across both patient-derived myofibroblasts and VSMCs. ResultsIndividuals with dominant negative (DN) variants in the MH2 protein interaction domain of SMAD3 exhibited a higher frequency of major events (66.7% vs. 44.0%, p=0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. Moreover, the age at the onset of the first major event was notably younger in individuals with DN variants in MH2, 35.0 years [IQR 29.0-47.0], compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (p=0.065). In functional assays, fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential observed in fibroblasts with HI SMAD3 variants. Additionally, HI SMAD3 variant VSMCs showed elevated SMA expression, while exhibiting altered expression of alternative MYH11 isoforms. Conversely, DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix (ECM) formation compared to control cell lines. These findings collectively indicate distinct functional consequences between DN and HI variants in SMAD3 across fibroblasts and VSMCs, potentially contributing to the observed differences in disease manifestation and age of onset of major events. ConclusionDistinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and evaluating SMA and MYH11 expression. Notably, myofibroblast differentiation seems to be a suitable alternative in vitro test system in comparison to VSMCs. Moreover, there is a notable trend of aortic events occurring at younger age in individuals with a DN SMAD3 variant in the MH2 domain, distinguishing them from those with a DN variant in the MH1 domain or a HI variant.

genetics↗

bioRxiv2023

Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells

Microfibrillar-associated protein 4 (MFAP4) is a 36-kDa extracellular glycoprotein with critical roles in human pathologies, including fibrosis in several organ systems, chronic obstructive pulmonary disease, and cardiovascular disorders. In elastic tissues such as arteries, lungs, and skin, MFAP4 associates with microfibrils and elastic fibres, which are the central extracellular fibres affected in thoracic aneurysms. MFAP4 directly interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. MFAP4 multimerisation represents a critical hallmark required for its physiological and pathological properties. However, molecular details and functional consequences of MFAP4 multimerisation are lacking. Here we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer with D2 point group symmetry, where two sets of homodimers constitute the top and bottom halves of each octamer. Each of the homodimers is linked together by an inter-molecular disulfide bond. An engineered C34S missense mutation in MFAP4 prevented disulfide-bond formation between monomers, but the mutant formed octamers similar to wild type MFAP4. The atomic model, built into the 3.55 [A] cryo-EM map, suggests that several salt-bridges are important for interactions within and between homodimers, while non-polar interactions are important for octamer halves to assemble. In the absence of calcium, MFAP4 dissociates into tetramers, representing the top/bottom halves of the octamers. Binding studies with elastogenic proteins, including fibrillin-1, tropoelastin, LTBP4, and small fibulins showed that MFAP4 has multiple surfaces for protein-protein interactions, which depend upon the higher-order assembly of MFAP4. While the disulfide-bond mediated by C34S contributes little to those protein interactions, it modulated cell interaction. When MFAP4 forms assemblies with fibrillin-1, it abrogates MFAP4 interactions with cells. Overall, the study provides detailed molecular structure-function relationships of MFAP4 interactions with elastogenic proteins and cells.

cell biology↗

bioRxiv2021

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet specific transition mechanisms ensuring ECM proteostasis and their regulatory impact are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of mouse Adamts6 and Adamts10 mutant embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics identification of ADAMTS6-cleaved sites, demonstrates a transcriptionally adapted system for fibrillin-2 proteolysis that contributes to postnatal fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development resulting from a drastic reduction of aggrecan, cartilage link protein and impaired BMP, but not TGF{beta} signaling in cartilage. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of these defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

developmental biology↗

bioRxiv2019

SLC2A knockout mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicate GLUT10 in transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations do not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in rodents. Gulo;Slc2a10 knock-out mice show mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 knock-out mice do not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. TGF{beta} signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

genetics↗