Biology subjects

Krahn, M. P.

Publications and source records attributed to Krahn, M. P..

5 recordsLinked to original sources

bioRxiv2026

Molecular orchestration of global actin remodelling by INF2

The actin cytoskeleton rapidly reorganizes in response to intracellular calcium signals, driving cellular morphogenesis and wound healing. Among actin regulators, the formin INF2 uniquely mediates the "Calcium-mediated Actin Reset" (CaAR) reaction, orchestrating transient and global actin remodeling upon calcium influx. Excessive INF2 activity is linked to kidney and neuronal diseases, underscoring the need for its tight control. Combining live cell imaging with single molecule tracking, biochemistry and structural analysis we discover that INF2 activity is tightly controlled by two interlinked mechanisms: canonical intramolecular autoinhibition and binding of the INF2 N-terminus to the side of actin filaments. Side-binding limits actin elongation and supports re-establishment of autoinhibition. Disruption of this negative feedback prolongs INF2 activity, affecting plasma membrane organization and repair as well as transcriptional control. Our findings uncover a novel product-inhibition mechanism that limits INF2 function and offer important insight into disease mechanisms linked to actin dysregulation.

cell biology↗

bioRxiv2023

Elucidating the Membrane Binding Process of the LKB1 C-terminus via Atomistic Simulations: Dynamic Interplay of Anionic Lipids and the Polybasic Region

Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase LKB1 - a serine/threonine kinase, relevant for cell proliferation and cell polarity. LKB1 itself becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of phosphatidylcholin (POPC) and phosphatidic acid (PA) and cell culture experiments. Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in a non-monotonous changes in the structure of the protein as well as of the membrane, in particular accumulation of PAs and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of the protein-membrane binding of LKB1. These results can be likely transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.

biophysics↗

bioRxiv2023

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved Crumbs (CRB) polarity complex, which consists of the core components CRB3a, PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. To address the role of PALS1 at the cellular level, we generated PALS1 knockout MDCKII cell lines using the CRISPR/Cas9 system. The loss of PALS1 resulted in increased paracellular permeability indicative of an epithelial barrier defect. This barrier defect was associated with a redistribution of several tight junction-associated proteins from bicellular cell-cell contacts to tricellular junctions. The regulation of tight junction protein localization at bicellular junctions by PALS1 was dependent on its interaction with PATJ. Together, our data uncover a critical role of PALS1 in the correct positioning of tight junction proteins to bicellular junctions.

cell biology↗

bioRxiv2021

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

cell biology↗

bioRxiv2019

A deregulated stress response underlies distinct INF2 associated disease profiles

Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Here we systematically characterize a large panel (>50) of autosomal dominant mutants of the actin regulator inverted formin 2 (INF2) that have been reported to cause the podocytic kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot Marie-Tooth disease (CMT). We found that INF2 mutations lead to deregulated activation of the formin and a constitutive stress response in cultured cells, primary patient cells and Drosophila nephrocytes. Using quantitative live-cell imaging we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. Furthermore, we could clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and CMT. Our results indicate that cellular profiling of disease-associated mutations can contribute substantially to sequence-based phenotype predictions.

cell biology↗