Matthias Preller

Background: Class I myosins are essential mediators of membrane–cytoskeleton interactions that support key cellular processes such as endocytosis, secretion, intracellular trafficking, and mitosis. However, the mechanisms driving isoform-specific targeting to membrane domains enriched in signaling lipids as well as their stage-dependent recruitment to mitotic structures during cell division remain poorly defined. Methods and approach: Using Dictyostelium discoideum as a highly phagocytic cell model, we demonstrate that long-tailed myosin-1 isoforms (myosin-1B, -1C, and − 1D) exhibit distinct lipid and cytoskeletal binding profiles shaped by their modular tails and variations within the phosphoinositide binding motif. Homology-based structural modelling of the PH-like lipid binding domain within the TH1 sequence, combined with molecular docking explains their differential lipid affinities. Kinetic equilibrium modelling with quantitative data suggests these differences enable cooperative or competitive isoform localization within cells providing a mechanism for temporally controlled recruitment of the myosins in response to dynamic changes in membrane composition and expression profiles. These biochemical insights are corroborated by confocal live-cell imaging, which reveals phosphoinositides-dependent localization dynamics and isoform-specific targeting of the myosins during vegetative growth and mitotic progression. Results: Myosin-C exhibits phosphoinositide binding preferences nearly reciprocal to those of myosin-1D, especially between mono- and triple phosphorylated phosphoinositides, and shows the strongest tail-mediated, ATP-independent actin binding. Myosin-1B, in contrast, displays low affinity for monophosphorylated phosphoinositides, intermediate actin binding ability, and no microtubule interaction. The comparable affinities of all three myosins for PI(3,5)P₂ and PI(4,5)P₂, the major PIP species at the cell cortex, facilitate their accumulation at membrane protrusions. Live-cell imaging confirms that myosin-1D preferentially associates with PI(3,4,5)P₃- and PI(3)P-enriched endosomes during macropinocytosis and phagocytosis, consistent with its higher binding affinity for these phosphoinositides. Conversely, myosin-1C localization is governed by both actin and phosphoinositides, enabling a rapid dissociation from early endosomes to retarget the cortex and accumulate at actin-rich phagocytic cup tips. Upon mitotic entry, myosin-1D, similar to myosin-1C, redistributes from endosomal compartments to the mitotic apparatus, where it decorates membrane-enclosed nuclear chromatin masses through its TH1 domain and later associates with spindle pole microtubules. This contrasts with myosin-1C, which selectively targets spindle microtubules throughout mitosis, reflecting its stronger microtubule-binding affinity. Inhibition of PI3-kinase disrupts membrane recruitment of both isoforms, confirming their phosphoinositide-dependent localization. These findings reveal an isoform-specific mechanism underlying myosin-1 targeting during endocytosis and mitosis. Conclusion: Collectively, these findings establish a phosphoinositide- and cytoskeleton-guided mechanism that governs myosin-1 isoform-specific functions, providing new insights into how motor proteins interpret complex lipid and cytoskeletal cues to regulate membrane remodelling and cytoskeletal dynamics across cellular states.

Amide synthases catalyze the formation of macrolactam rings from aniline-containing polyketide-derived seco-acids as found in the important class of ansamycin antibiotics. One of these amide synthases is the geldanamycin amide synthase GdmF, which we recombinantly expressed, purified and studied in detail both functionally as well as structurally. Here we show that purified GdmF catalyzes the amide formation using synthetically derived substrates. The atomic structures of the ligand-free enzyme and in complex with simplified substrates reveal distinct structural features of the substrate binding site and a putative role of the flexible interdomain region for the catalysis reaction.

Aberrant Ras homologous (Rho) GTPase signalling is a major driver of cancer metastasis, and GTPase-activating proteins (GAPs), the negative regulators of RhoGTPases, are considered promising targets for suppressing metastasis, yet drug discovery efforts have remained elusive. Here, we report the identification and characterization of adhibin, a synthetic allosteric inhibitor of RhoGAP class-IX myosins that abrogates ATPase and motor function, suppressing RhoGTPase-mediated modes of cancer cell metastasis. In human and murine adenocarcinoma and melanoma cell models, including three-dimensional spheroid cultures, we reveal anti-migratory and anti-adhesive properties of adhibin that originate from local disturbances in RhoA/ROCK-regulated signalling, affecting actin-dynamics and actomyosin-based cell-contractility. Adhibin blocks membrane protrusion formation, disturbs remodelling of cell-matrix adhesions, affects contractile ring formation, and disrupts epithelial junction stability; processes severely impairing single/collective cell migration and cytokinesis. Combined with the non-toxic, non-pathological signatures of adhibin validated in organoids, mouse and Drosophila models, this mechanism of action provides the basis for developing anti-metastatic cancer therapies.

The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na⁺ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTR_inh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTR_inh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca²⁺ release-activated Ca²⁺ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

The author wishes to make the following correction to this paper

Microarray-based experiments revealed that thyroid hormone triiodothyronine (T3) enhanced the binding of Cy5-labeled ATP on heat shock protein 90 (Hsp90). By molecular docking experiments with T3 on Hsp90, we identified a T3 binding site (TBS) near the ATP binding site on Hsp90. A synthetic peptide encoding HHHHHHRIKEIVKKHSQFIGYPITLFVEKE derived from the TBS on Hsp90 showed, in MST experiments, the binding of T3 at an EC₅₀ of 50 μM. The binding motif can influence the activity of Hsp90 by hindering ATP accessibility or the release of ADP.

The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with P_i release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered P_i release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

Several small molecule effectors of myosin function that target the motor domains of myosin classes I, II, V, and VI have been identified. Four distinct binding sites in the myosin motor domain have been reported with unique properties and mechanisms of action. This chapter describes the structural basis and activities of known small molecule effectors that allosterically target the myosin motor domain.