Biology subjects

Busch, K. B.

Publications and source records attributed to Busch, K. B..

9 recordsLinked to original sources

bioRxiv2026

A dynamic displacement mechanism drives protein import into mitochondria

Most mitochondrial proteins are produced in the cytosol and imported through the translocase of the outer mitochondrial membrane (TOM) to reach their final destination. Although this protein entry gate has been structurally characterized, it remains unclear how precursor proteins are handed off from the cytosolic receptor domains to the translocation pore. Here we show that the cytosolic domain of Tom22--traditionally viewed as the central TOM receptor--acts not as a structured scaffold but as a largely disordered, flexible segment that plays an active role in precursor transfer. Atomic-level structural techniques and in vivo experiments identified a conserved short linear motif that forms a transient !-helical element within this disordered domain. By binding to the canonical precursor protein binding sites of the receptors Tom20 and Tom70, this critical -helical segment acts as a precursor protein displacement element (PPDE). This competitive interaction facilitates the release of preproteins directly above the import pore, and thereby drives translocation across the outer mitochondrial membrane. These findings reveal that flexibility, rather than rigid structure, underlies the central transfer step of mitochondrial outermembrane protein translocation. Our results point to a versatile mechanism for ligand displacement in chaperone, receptor, and transport systems that must balance selective binding with efficient release.

cell biology↗

bioRxiv2025

Multi-scale mitochondrial cristae remodeling links Opa1 downregulation to reduced OXPHOS capacity in aged hearts

Aging is closely associated with cardiovascular diseases, the leading cause of mortality worldwide. Mitochondrial dysfunction is a hallmark of cardiovascular aging because it generates most of the hearts ATP at the cristae, specialized sub-compartments where OXPHOS takes place. In this study, we used multiple-scale electron microscopy approaches to evaluate age-related mitochondrial and cristae ultrastructural alterations in human and mouse hearts. We found that aged patients hearts displayed reduced cristae density as seen by TEM, even before any significant decline in the expression of cristae-shaping proteins. Similarly, a multi-scale approach that included SBF-SEM and TEM showed that in aged mices hearts cristae undergo ultrastructural remodeling processes, resulting in a decrease in cristae density and width. Electron tomography suggests an apparent decline in cristae connectivity, and an increase in fenestration size. These changes were linked to Opa1 downregulation, accompanied by reduced OXPHOS maximal respiration, but unrelated to alterations in the levels of OXPHOS core subunits and ATP synthase assembly. Altogether, this indicates that alterations in cristae structure alone are sufficient to impair oxidative metabolism, which highlights its potential as an early signal of cardiac aging, even before noticeable changes in mitochondrial morphology occur.

cell biology↗

bioRxiv2024

Decreased ATP synthase activity is linked to altered spatiotemporal organisation of ATP Synthase in a cellular cardiomyocyte senescent model

Heart disease is the leading cause of death in the elderly population and the heart is a highly energy-consuming tissue. Aging-related heart failure is often driven by energy depletion in cardiomyocytes (CM), which rely on their abundant, cristae-dense mitochondria for ATP production. ATP synthase, localized along the cristae rims, plays a critical role in energy conversion, but the connection between its organization and function remains unclear. Here, we explored the spatiotemporal organization of ATP synthase in senescent CM at the level of individual complexes. Using single-molecule localization and tracking microscopy, we observed reduced enzyme mobility within the cristae, coinciding with decreased ATP synthase activity, despite a stable resting mitochondrial membrane potential. This reduction in activity was independent of changes in ATP synthase expression or dimerization. Electron tomography revealed an increased prevalence of curved inner membranes and fenestrated cristae in senescent CM, explaining the reduced enzyme mobility. Senescent CM displayed irregular autonomous and paced beating patterns. These abnormalities suggest that impaired cardiac function is directly driven by disrupted energy metabolism, rooted in the suboptimal organization and function of ATP synthase in altered cristae.

cell biology↗

bioRxiv2024

TC10 differently controls the dynamics of Exo70 in growth cones of cortical and hippocampal neurons

The exocyst is an octameric protein complex that acts as a tether for GOLGI-derived vesicles at the plasma membrane during exocytosis. It is involved in membrane expansion during axonal outgrowth. Exo70 is a major subunit of the exocyst complex and is controlled by TC10, a Rho family GTPase. How TC10 affects the dynamics of Exo70 at the plasma membrane is not well understood. There is also evidence that TC10 controls Exo70 dynamics differently in nonpolar cells and axons. To address this, we used super-resolution microscopy to study the spatially resolved effects of TC10 on Exo70 dynamics in HeLa cells and the growth cone of cortical and hippocampal neurons. We generated single particle localization and trajectory maps and extracted mean square displacements, diffusion coefficients, and alpha coefficients to characterize Exo70 diffusion. We found that the diffusivity of Exo70 was different in nonpolar cells and in the growth cone of neurons. TC10 stimulated the mobility of Exo70 in HeLa cells, but decreased the diffusion of Exo70 in the growth cone of cortical neurons. In contrast to cortical neurons, TC10 overexpression did not affect the mobility of Exo70 in the axonal growth cone of hippocampal neurons. These data suggest that mainly exocyst tethering in cortical neurons was under the control of TC10.

biophysics↗

bioRxiv2024

Tagging of Exo70 at the N-terminus compromises its assembly with the exocyst complex and changes its spatiotemporal behavior at the plasma membrane

The vesicle-tethering exocyst complex is a key regulator of cell polarity. The subunit Exo70 is required for the targeting of the exocyst complex to the plasma membrane. While the N-terminus of Exo70 is important for its regulation by GTPases, the C-terminus binds to PI(4,5)P2 and Arp2/3. Here, we compare N- and C-terminal tagged Exo70 with respect to subcellular localization, dynamics and function in cell membrane expansion. Using high-resolution imaging, we determined the spatial distribution and dynamics in different sub-compartments of un-polarized and polarized cells. With lattice light-sheet microscopy, we show that HaloTag-Exo70, but not Exo70-HaloTag, promotes the outgrowth of filopodia-like structures from the axon of hippocampal neurons. Fluorescence lifetime imaging of sfGFP-Exo70 and molecular modeling results suggest that the assembly of sfGFP-Exo70 with the exocyst complex is reduced. This is supported by single particle tracking data showing higher mobility of N- than C-terminal tagged Exo70 at the plasma membrane. The distinct spatiotemporal properties of N-terminal tagged Exo70 were correlated with pronounced filopodia formation in unpolarized cells and neurons, a process that is less reliant on exocyst complex formation. We therefore propose that N-terminal tagging of Exo70 shifts its activity to processes that are less exocyst-dependent. Why it mattersIn the life sciences, the high-resolution visualization of processes in living cells is of great interest. To tag proteins, they are fused with fluorescent proteins, usually at the N- or C-terminus of the amino acid sequence. We show here that the position of the tag alters the function and interaction of Exo70, a polypeptide involved in vesicle fusion, but also in membrane bending and expansion. Using state-of-the-art microscopic techniques, single particle localization and tracking, fluorescence lifetime imaging microscopy and co-localization in combination with modeling, we conclude that N-terminal tagging of Exo70 impairs its assembly with the exocyst complex and instead promotes the interaction of free Exo70 with the actin skeleton, which favors controlled membrane expansion into filipodia.

biophysics↗

bioRxiv2024

Mdivi-1 affects neuronal activity by inhibition of Complex I and respiratory supercomplex assembly

Several human diseases, including cancer and neurodegeneration, are associated with excessive mitochondrial fragmentation. In this context, mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic to block the fission-related protein dynamin-like protein-1 (Drp1). Recent studies suggest that Mdivi-1 interferes with mitochondrial bioenergetics. Here we show that the molecular mechanism of Mdivi-1 is based on inhibition of complex I at the IQ site. This leads to the destabilization of complex I, impairs the assembly of N- and Q-respirasomes and is associated with increased ROS production. The result is a reduced efficiency of ATP generation. Second, the calcium homeostasis of cells is impaired, which severely affects the electrical activity of neurons. Given the results presented here, a potential therapeutic application of Mdivi-1 is challenging because of its impact on synaptic activity. Similar to the Complex I inhibitor rotenone, Mdivi-1 may lead to neurodegenerative effects in the long term. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/577160v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1bffc82org.highwire.dtl.DTLVardef@15ae3b5org.highwire.dtl.DTLVardef@1ba3a5org.highwire.dtl.DTLVardef@94cfc0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIMdivi-1 inhibits respiratory complex I at the IQ-site C_LIO_LIInhibition destabilizes complex I and reduces supercomplex formation C_LIO_LIMitochondrial ATP levels decrease C_LIO_LICa2+ metabolism is affected C_LIO_LINeuronal activity is compromised C_LI

cell biology↗

bioRxiv2022

LUBAC assembles a signaling platform at mitochondria for signal amplification and shuttling of NF-ĸB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-B pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-B to the nucleus. TNF induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-B is locally activated and transported to the nucleus by mitochondria, resulting in an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN.

cell biology↗

bioRxiv2021

pH profiling of mitochondrial sub-compartments reveals that IF1 is essential to prevent ATP hydrolysis under OXPHOS conditions

The classical view of oxidative phosphorylation is that a proton motive force PMF generated by the respiratory chain complexes fuels ATP synthesis. Under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected the two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions in detail. pH profiles of mitochondrial sub-compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH-sensor directly at F1 and FO of ATP synthase, complex IV and in the matrix. Our results clearly show that ATP synthase activity is substantially controlling the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible {Delta}pH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/430746v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1a975eforg.highwire.dtl.DTLVardef@7ddb49org.highwire.dtl.DTLVardef@c7a128org.highwire.dtl.DTLVardef@2559e8_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIThe {Delta}pH along and across the inner mitochondrial membrane is not homogeneous C_LIO_LIThe proton motive force at cristae tips is controlled by F1 FO ATP synthase C_LIO_LIUnder OXPHOS conditions, the pH difference between FO and F1 of active ATP synthase is almost negligible (1.2 proton vs. 1 proton equivalent) C_LIO_LIIF1 is required to prevent the onset of ATP hydrolysis under OXPHOS conditions C_LI

cell biology↗

bioRxiv2018

F1FO ATP synthase responds to glycolysis inhibition by localization into the inner boundary membrane

Mitochondrial F1F0 ATP synthase is the key enzyme to fuel the cell with essential ATP. Strong indications exist that the respiratory chain and the ATP synthase are physically separated within cristae. How static this organization is, is largely unknown. Here, we investigated the effect of substrate restriction on mitochondrial respiration and the spatio-temporal organization of ATP synthase. By superresolution microscopy, the localization and mobility of single labelled mitochondrial ATP synthase was determined in live cells. We found, that the ATP synthase under oxidative respiration displayed a clear localization and confined mobility in cristae. Trajectories of individual ATP synthase proteins show a perpendicular course to the longitudinal axis of the respective mitochondrion, exactly following the ultrastructure of cristae. When substrate for TCA cycle and respiration was limited, a significant proportion of ATP synthase localized from cristae to the inner boundary membrane, and only less mobile ATP synthase remained in cristae. These observations showing the plasticity of the spatio-temporal organisation of ATP synthase can explain why ATP synthase show interactions with proteins in distinct mitochondrial subcompartments such as inner boundary membrane, cristae junctions and cristae.

cell biology↗