Biology subjects

Pondman, K.

Publications and source records attributed to Pondman, K..

3 recordsLinked to original sources

bioRxiv2024

The human complement factor B-C3b complex: Investigation of the interaction using C3b bound to thiol-Sepharose

Factor B, a serine protease proenzyme and part of the complement system, binds to other complement proteins such as C3b and properdin. While it is known to be activated by factor D, other interactions that interfere with the binding between Factor B and C3b are less well-understood. We attached C3b to a thiol Sepharose via its free SH group and conducted a competition assay employing 125I-labelled factor B to study competitive binding. Two anti-C3d monoclonal antibodies (F49b and 4C2) were found to partially inhibit the binding of Factor B to C3b. The inhibitory effects of these monoclonal antibodies were found to be dose-dependent. 4C2 was found to have a maximum inhibition of 50%, while the maximum inhibition by F49b was [~]40%. In contrast, two anti-C3c monoclonal antibodies (F39b and F20b) were able to enhance the formation of the factor B-C3b complex in a dose-dependent manner. Competition binding studies with isolated C3 fragments further supported the involvement of the C3d region in factor B binding, as C3d inhibited the binding of factor B to C3b completely. Furthermore, additional competition binding studies conducted with each of the three domains of factor B (Ba, vWF and SP) demonstrated that each domain independently inhibited the binding of intact factor B to C3b by [~]50%.

immunology↗

bioRxiv2023

3D single-molecule super-resolution imaging of microfabricated fractal substrates for cell culture and self-referenced imaging

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity, as well as axial calibration issues. In this work, we address this issue by leveraging the self-referenced characteristics of fractal-like structures, which simultaneously modulate cell growth and serve as axial calibration tools. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as calibration tools for 3D super-resolution microscopy. We use their intrinsic multiscale and self-referenced nature to simultaneously perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilize these substrates as a platform for high-resolution bioimaging. As proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with 3D SMLM assessment.

biophysics↗

bioRxiv2023

Activation of Complement by Human Fibrin Clots: involvement of C1q and factor H

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator including immune complexes. Factor H is regarded as the key downregulatory protein of the alternative pathway of complement. However, C1q and factor H both bind to target surfaces via charge distribution patterns. For few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation by such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are reported to recognize "foreign" or altered-self materials. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the classical pathway of complement and whether this is regulated by factor H. We show here that both C1q and factor H bind to fibrin formed in microtitre plates as well as fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the classical pathway activation induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

immunology↗