Biology subjects

Mahlandt, E. K.

Publications and source records attributed to Mahlandt, E. K..

8 recordsLinked to original sources

bioRxiv2022

iLID-antiGFP-nanobody is a flexible targeting strategy for recruitment to GFP-tagged proteins

Optogenetics is a fast-growing field, that applies light-sensitive proteins to manipulate cellular processes. A popular optogenetics tool is the improved light-induced dimer (iLID). It comprises two components, iLID and SspB, which heterodimerize upon illumination with blue light. This system is often used to recruit proteins to a specific subcellular location, e.g. by targeting the iLID to the plasma membrane. The targeting requires modification of the iLID with a targeting sequence. To skip the modification of the iLID and use existing GFP fusion as targets, we fuse an antiGFP nanobody to the iLID. We show that the antiGFP nanobody is able to locate iLID to GFP-tagged proteins. Plus, the light-dependent recruitment of SspB to iLID, localized by the antiGFP nanobody to a GFP-tagged protein, is still functioning efficiently. This approach increases flexibility, enabling the recruitment of any GFP-tagged protein, without the necessity of protein engineering.

cell biology↗

bioRxiv2022

Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated to allow the passage of essential molecules like oxygen, carbon-dioxide, water, ions, and nutrients. The vascular endothelial barrier is defined by cell-cell contacts through adherens and tight junctions. To further investigate the signaling in the endothelium that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. Rho GTPase signaling is confined in space and time. To manipulate the signaling in a temporal and spatial manner we applied optogenetics. Guanine exchange factor (GEF) domains from ITSN1, TIAM1 and p63RhoGEF, activating Cdc42, Rac and Rho respectively, were integrated into the optogenetic recruitment tool iLID. This tool allows for activation at the subcellular level in a reversible and non-invasive manner and thereby to recruit a GEF to local areas at the plasma membrane, enabling the local activation of specific Rho GTPases. The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting Opto-RhoGEFs were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell-size, -roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool i.e. Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

cell biology↗

bioRxiv2022

A systematic, cell-based comparison of G-protein binding domains for their potential as localization-based, genetically encoded biosensors of Cdc42 or Rac activity

Rac and Cdc42 are Rho GTPases which regulate the formation of lamellipoda and filopodia and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for either Rac or Cdc42. We compared their (i) ability to bind the constitutively active Rho GTPases, (ii) specificity for Rac and Cdc42 and (iii) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1 we found a sensor candidate with low relocation efficiency. For Cdc42 we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. The characterization and optimization of relocation sensors will broaden their application and acceptance.

cell biology↗

bioRxiv2022

ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia

Invadopodia formation is regulated by Rho GTPases. However, the molecular mechanisms that control Rho GTPase signaling at invadopodia remain poorly understood. Here, we have identified ARHGAP17, a Cdc42-specific RhoGAP, as a key regulator of invadopodia in breast cancer cells and by RhoGAPs characterized a novel ARHGAP17-mediated signaling pathway that controls the spatiotemporal activity of Cdc42 during invadopodia turnover. Our results show that during invadopodia assembly, ARHGAP17 localizes to the invadopodia ring and restricts the activity of Cdc42 to the invadopodia core, where it promotes invadopodia growth. Invadopodia disassembly starts when ARHGAP17 translocates from the invadopodia ring to the core, in a process that is mediated by its interaction with the Cdc42 effector CIP4. Once at the core, ARHGAP17 inactivates Cdc42 to promote invadopodia disassembly. Our results in invadopodia provide new insights on the coordinated transition between the activation and inactivation of Rho GTPases.

cell biology↗

bioRxiv2021

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a novel sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. A new engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/449214v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@12beb07org.highwire.dtl.DTLVardef@5337f1org.highwire.dtl.DTLVardef@17db68forg.highwire.dtl.DTLVardef@5ca499_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Visualizing and quantifying data from timelapse imaging experiments

One obvious feature of life is that it is highly dynamic. The dynamics can be captured by movies that are made by acquiring images at regular time intervals, a method that is also known as timelapse imaging. Looking at movies is a great way to learn more about the dynamics in cells, tissue and organisms. However, science is different from Netflix, in that it aims for a quantitative understanding of the dynamics. The quantification is important for the comparison of dynamics and to study effects of perturbations. Here, we provide detailed processing and analysis methods that we commonly use to analyze and visualize our timelapse imaging data. All methods use freely available open-source software and use example data that is available from an online data repository. The step-by-step guides together with example data allow for fully reproducible workflows that can be modified and adjusted to visualize and quantify other data from timelapse imaging experiments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/432684v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@d04086org.highwire.dtl.DTLVardef@3c304borg.highwire.dtl.DTLVardef@186bf56org.highwire.dtl.DTLVardef@17bbd6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development, cell migration and wound healing. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes, in real time with subcellular resolution in living cells and tissue. Until now, relocation RhoA biosensors seem to only be useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and RhoA binding peptides, on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded, fluorescent biosensors for detecting endogenous RhoA activity. This enables a broader application of RhoA relocation biosensors, which was demonstrated by using the improved biosensor to visualize RhoA activity, during cell division, during random migration, at the Golgi membrane and induced by G protein-coupled receptor signaling. Due to the improved avidity of the new biosensors for RhoA activity, cellular processes regulated by RhoA can be better understood. O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/430250v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1847c24org.highwire.dtl.DTLVardef@f22c92org.highwire.dtl.DTLVardef@14c4aa8org.highwire.dtl.DTLVardef@1b9657d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

cell biology↗