Biology subjects

Dehmelt, L.

Publications and source records attributed to Dehmelt, L..

9 recordsLinked to original sources

bioRxiv2026

Selective covalent-allosteric tools to dissect Akt2

The protein kinase Akt and its isoforms play a crucial role in various diseases. Unique functions of the individual isoforms (Akt1, Akt2, Akt3) might be essential for survival in malignancies. Particularly for Akt2, it was reported that a knock-out led to diabetic phenotype and might be correlated with clinically adverse hyperglycemic effects observed in pan Akt-treatment. Enduring failure of Akt inhibitors in the clinic indicates the necessity for a thorough understanding of the underlying biology, preferably by using highly isoform-selective small molecules. Here we report the structure-guided development of Akt2-selective covalent-allosteric probe molecules, that can be successfully modified within a complex environment using biorthogonal chemistry. Thus, enabling first Akt2-specific pull-down studies and the use in functional studies, such as selective fluorescent labeling in cellular systems. These chemical probes expand our toolbox to dissect the critical questions of Akt2s function in health and disease, thereby paving the way for novel therapeutic strategies based on thorough mechanistic insights.

Cancer Biology↗

bioRxiv2026

Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

During cell division, multiple guanine nucleotide exchange factors (GEFs), including the mitotic regulator Ect2 and Lbc-type GEFs, regulate the dynamics of RhoA activity and actomyosin contractility. In interphase, Lbc-type GEFs are predominantly cytosolic and are transiently recruited to the plasma membrane via a Rho-dependent positive-feedback mechanism that generates stochastic pulses of Rho activity and cortical contractions. In contrast, during interphase, Ect2 is primarily sequestered in the nucleus away from the cytoplasm and the plasma membrane. Following nuclear envelope breakdown in mitosis, Ect2 is released into the cytosol and constitutively associates with the plasma membrane independently of Rho via a C-terminal polybasic cluster. Here, we mimicked this mitotic state by expressing a constitutively active Ect2 variant that is lacking its nuclear localization signal in interphase, to examine its interplay with Lbc-stimulated pulsatile Rho dynamics. We found that active cytosolic Ect2 suppressed pulsatile dynamics and promoted peripheral enrichment of Rho activity and myosin. Pulse suppression required Ect2 catalytic activity, Rho-dependent positive feedback, and constitutive plasma membrane association. In contrast, an Ect2 variant that was lacking constitutive plasma membrane association via the polybasic cluster, was not only incapable of pulse suppression but instead even stimulated strong pulsatile Rho dynamics. Together, our results identify constitutive plasma membrane association as a key distinction between Lbc-type GEFs and Ect2, and demonstrate that this property enables Ect2 to suppress pulsatile Rho activity dynamics, thereby promoting a stable, non-pulsatile contractile signaling regime during mitosis.

cell biology↗

bioRxiv2025

Logic-gating the HaloTag system with Conditional-Halo-ligator 'CHalo' reagents

HaloTag proteins spontaneously ligate onto any chemical reagent featuring a chloroalkane motif (CA). We introduce the conditional CHalo motif, which ligates to HaloTag only after uncaging by light or enzymes. (1) Photo-triggered CHalo fluorogenic reagents allow spatiotemporally-specific labeling; (2) photo-triggered CHalo heterodimerisers can photocontrol protein recruitment; and (3) enzyme-triggered CHalo reagents can durably record diverse enzyme activities, and multiplexing them should allow quantitative ratiometric recording of multiple activities in parallel. CHalo thus permits manifold extensions to the HaloTag technology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/676741v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d18cd6org.highwire.dtl.DTLVardef@175694forg.highwire.dtl.DTLVardef@154ffd5org.highwire.dtl.DTLVardef@167fec2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Rnd3 regulates cell morphodynamics by spatial restriction of cell contraction signaling

Cell migration is enabled by dynamic changes in cell shape, which are controlled by spatio-temporal activity patterns of the Rho GTPases Rac1 and RhoA. Classical models proposed that these activity patterns are generated by mutual inhibition between the front signal Rac1 and the back signal RhoA, leading to opposing gradients that define the direction of cell migration. However, direct measurements of signal crosstalk showed that Rac1 can activate RhoA, which is incompatible with mutual inhibition. Furthermore, opposing Rac1 and RhoA gradients generated by mutual inhibition would need to overlap at least partially in the cell center. In contrast, both Rac and Rho activities were largely absent in the cell center, and both found to be highly localized near the cell edge in the cell periphery. Here, we hypothesized that these spatio-temporal Rho GTPase activity patterns are generated by the mutual inhibition between RhoA and the unconventional Rho family member Rnd3. Using rapid, optogenetic and chemical perturbations, we confirmed this mutual inhibitory crosstalk. However, we found that this crosstalk does not lead to the expected mutually exclusive spatio-temporal patterns of Rnd3 activity and cell retraction in spontaneously migrating, unperturbed cells. Instead, Rnd3 activity was even slightly elevated during cell retraction and was surprisingly depleted during cell protrusion. We discovered that this depletion is caused by the inhibition of Rnd3 by Rac1 activity, and that this newly identified inhibition is much more pronounced compared to the inhibition of Rnd3 by Rho. By combining rapid optogenetic perturbations with pharmacological manipulations, we found that Rac1 inhibition by Rnd3 is mediated by p21-activated kinases (PAKs). Investigations into the function of Rnd3 showed that it is required for the tight spatio-temporal regulation of the cell contraction/retraction signal Rho, and that it prevents ectopic, highly dynamic, spontaneous Rho activity pulses within the whole cell attachment area. Interestingly, protrusion-retraction dynamics were also severely inhibited in the absence of Rnd3, and its overexpression stimulated this process. Taken together, we show that Rac and not Rho acts as the major Rnd3 inhibitor in cells. Furthermore, our findings support a mechanism, in which Rnd3 acts as a global inhibitor of Rho that spatially restricts Rho activity to regions near the edge of migrating cells.

cell biology↗

bioRxiv2025

The ROCK1 PH domain interacts with active Rho to transduce cell contraction signals

The spatio-temporal regulation of cell contraction is crucial for numerous biological processes. In particular, contraction pulses in the cell cortex contribute to mechanotransduction and tissue rearrangements during embryonic development. We previously identified a signaling network that generates mechanosensitive contraction pulses in adherent mammalian cells via positive and negative feedback regulation of the small GTPase RhoA. Our investigations into the molecular mechanism of this process revealed surprising observations that challenge prevailing models of ROCK1 regulation. In particular, we identified a novel RhoA binding site in the ROCK1 PHC1 tandem domain that is sufficient for dynamic recruitment leading to increased Rho activity within subcellular regions of the cell cortex. AlphaFold-guided mutagenesis supports a direct interaction between these molecules. Functional investigations show that the PHC1 domain is required for efficient recruitment to active Rho, and that it plays a role in the transduction of Rho activity via ROCK1 to Myosin II activation. Based on the newly identified Rho binding site at the C-terminus of ROCK1, we propose a model for ROCK1 activation, which can resolve inconsistencies between previous biochemical and structural studies.

cell biology↗

bioRxiv2025

Non-muscle Myosin II acts as a negative feedback mediator to control cell contraction dynamics

Local cell contraction dynamics play a crucial role in tissue and cell morphogenesis. Contractions near the cell edge drive highly dynamic cell shape changes during cell migration and contraction pulses in central cell attachment areas are involved in mechanotransduction. Previously, we identified a signal network in adherent mammalian cells, that generates mechanosensitive contraction pulses, in which the cell contraction regulator Rho is controlled by fast positive feedback amplification via GEF-H1, and by a slow negative feedback that depends on actomyosin activity. However, the precise mechanism of this negative feedback in adherent cells, in particular if it is mediated via actin or Myosin-based components, was still unclear. Here, using numerical simulations of this system, we predicted that the cell contraction signal network dynamics are strongly inhibited both by inhibition and by constitutive activation of the actomyosin component Myosin-II. We confirmed these predictions experimentally by direct inhibition of Myosin-II and by activation via constitutively active ROCK1. Furthermore, constitutive activation of Myosin-II leads to an accumulation of Myosin-II next to the nuclei which spatially correlated with a corresponding shift of Rho activity dynamics from the cell center to the cell edge, showing that constant Myosin activation can spatially restrict cell contraction dynamics. Finally, light-induced rapid recruitment of ROCK1 to the plasma membrane strongly activated and recruited Myosin-II, and at the same time depleted Actin and inhibited Rho activity at the plasma membrane. We conclude that negative feedback in the cell contraction signal network of adherent mammalian cells is mediated by Myosin-II, and that actin does not act as the predominant inhibitor in this system.

cell biology↗

bioRxiv2025

Optogenetic stimulation of Lbc GEF-mediated Rho activity dynamics promotes cell invasion

Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression, however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model, in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs, and how they might promote tumor cell invasion. We propose a mechanism, in which increased cell contraction dynamics results in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.

cell biology↗

bioRxiv2025

Crosstalk between Rac and Rap GTPases in migrating cells

To enable effective cell migration, local cell protrusion has to be coordinated with local cell attachment. Here, we investigate spatio-temporal activity patterns of key regulators of cell protrusion and adhesion, the small GTPases Rac and Rap, in migrating cells. These analyses show that Rac activity correlates very tightly with instantaneous cell protrusion events, while the Rap activity stays elevated for prolonged time periods after protrusion and is also detectable before cell protrusion. Direct analysis of activity crosstalk in living cells via light-based perturbation methods revealed that Rap can efficiently activate Rac, however, reciprocal crosstalk from Rac to Rap was not detectable. These findings suggest that Rap plays an instructive role in the generation of cell protrusions by its ability to activate Rac. Furthermore, prolonged Rap activity suggests that this molecule also plays a role in maintenance or stabilization of cell protrusions. Indeed, morphological analysis of Rap1-depleted A431 cells revealed a significant reduction of the cell attachment area, suggesting that Rap stimulated cell adhesion might indeed stabilize newly formed protrusions. Taken together, our study suggests a mechanism, by which cell protrusion is coupled to cell adhesion via unidirectional crosstalk that connects the activity of the small GTPases Rap and Rac.

cell biology↗

bioRxiv2023

Crosstalk between Rac and Rho GTPase activity mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by a decrease in migration distance and an increase in migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.

cell biology↗