Biology subjects

Flemming, S.

Publications and source records attributed to Flemming, S..

7 recordsLinked to original sources

bioRxiv2024

Chemically induced dislocation and mutation of essential cytoskeletal proteins: A case study focusing on Coronin-A

Actin is one of the most highly conserved proteins--the actin cytoskeleton or actin-like structures are found in all kingdoms of life. In this work, Coronin-A, a central player involved in actin dynamics, is considered as an example to compare the effect of four mutation strategies on its function in Dictyostelium discoideum. Two distinct genetic knockout methods (selection-linked integration and classical gene disruption), RNAi knockdown, and sudden loss-of-function generated by chemically induced dislocation (CID) are compared and the cell lines are characterized according to actin dependent processes: appearance, development, and motility. These phenotypic features are affected to different degrees in the mutant cell lines, providing a basis to discuss the strengths and weaknesses of each approach. This comparative study highlights the advantages of the newly introduced selection-linked integration method to knock out genes over commonly used gene disruption, and for expressing a CID-based knock-sideways system under the native promotor of the protein of interest.

cell biology↗

bioRxiv2023

Identification of a Rabenosyn-5 like protein and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of P. falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasites food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the re-purposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5 and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC-uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.

microbiology↗

bioRxiv2022

The Kelch13 compartment is a hub of highly divergent vesicle trafficking proteins in malaria parasites

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in ART resistance. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites.

cell biology↗

bioRxiv2022

Soluble VE-cadherin disrupts endothelial barrier function via VE-PTP/RhoA signalling

AimIncreased levels of soluble Vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We tested whether sVE-cadherin is critically involved in the onset of endothelial barrier dysfunction. Methods and ResultsApplication of recombinant human sVE-cadherin (extracellular domains EC1-5) on human microvascular endothelial cells in vitro and in a rat model in vivo induced loss of endothelial barrier function and reduced microcirculatory flow. sVE-cadherinEC1-5 led to decreased localization of VE-cadherin at cell borders. Additionally, sVE-cadherinEC1-5 perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction. VE-PTP inhibitor AKB9778 blunted all sVE-cadherinEC1-5-induced effects in vitro and in vivo. Downstream effects involve VE-PTP-dependent RhoA activation which was attenuated by AKB9778. Rho-kinase inhibitor Y27632 blocked sVE-cadherinEC1-5-induced loss of endothelial barrier function. ConclusionsVE-cadherin disrupts endothelial barrier function by dismantling the VE-cadherin complex at cell borders via VE-PTP-dependent RhoA activation. This uncovers a novel pathophysiological role of sVE-cadherin in the context of endothelial barrier dysfunction in inflammation.

physiology↗

bioRxiv2022

Tyrosine kinase inhibitors trigger lysosomal damage-associated cell lysis to activate the NLRP3 inflammasome

Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs. One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.

immunology↗

bioRxiv2022

Versatile patterns in the actin cortex of motile cells: Self-organized pulses can coexist with macropinocytic ring-shaped waves

Self-organized patterns in the actin cytoskeleton are essential for eukaryotic cellular life. They are the building blocks of many functional structures that often operate simultaneously to facilitate, for example, nutrient uptake and movement of cells. However, to identify how qualitatively distinct actin patterns can coexist remains a challenge. Here, we use bifurcation theory to reveal a generic mechanism of pattern coexistence, showing that different types of wave patterns can simultaneously emerge in the actin system. Our theoretical analysis is complemented by live-cell imaging experiments revealing that narrow, planar, and fast-moving excitable pulses may indeed coexist with ring-shaped macropinocytic actin waves in the cortex of motile amoeboid cells.

biophysics↗

bioRxiv2021

An unusual trafficking domain in MSRP6 defines a complex needed for Maurer's clefts anchoring and maintenance in P. falciparum infected red blood cells

Intracellular malaria blood stage parasites remodel their host cell, a process essential for parasite survival and a cause of pathology in malaria infections. Host cell remodeling depends on the export of different classes of exported parasite proteins into the infected red blood cell (RBC). Here we show that members of a recently discovered group of difficult to predict exported proteins harbor an N-terminal export domain, similar to other classes of exported proteins, indicating that this is a common theme among all classes of exported proteins. For one such protein, MSRP6 (MSP-7 related protein 6), we identified a second, untypical export-mediating domain that corresponded to its MSP7-like region. In addition to its function in export, this domain also mediated attachment to the Maurers clefts, prominent parasite-induced structures in the host cell where MSRP6 is located. Using BioID with the Maurers clefts attachment domain of MSRP6 to identify interactors and compartment neighbors in live parasites we discovered a novel complex of proteins at the Maurers clefts. We show that this complex is necessary for the anchoring and maintaining the structural integrity of the Maurers clefts. The Maurers clefts are believed to be involved in the transport of the major virulence factor PfEMP1 to the host cell surface where it mediates cytoadherence of infected RBCs to endothelial cells, a main reason for the importance of host cell modifications for parasite virulence in the human host. Taking advantage of MSRP6 complex mutants and IT4 parasites that we modified to express only one specific PfEMP1 we find that abolishing Maurers clefts anchoring was neither needed for PfEMP1 transport to the host cell surface nor for cytoadherence. Altogether, this work reveals parasite proteins involved in Maurers clefts anchoring and maintenance and unexpectedly finds that these functions seem to be dispensable for virulence factor transport and surface display.

microbiology↗