Biology subjects

Morgan, T. E.

Publications and source records attributed to Morgan, T. E..

9 recordsLinked to original sources

bioRxiv2026

Mechanism and regulation of Bim1 interactions withthe S. cerevisiae outer kinetochore Ndc80 complex

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an alpha-helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

molecular biology↗

bioRxiv2026

A GABARAP-PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation

Macroautophagy/autophagy is a cellular process enabling degradation of intracellular components during starvation. In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized within a brief time remains elusive. A protein complex central to the phagophore initiation is the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits a variety of downstream proteins, among which is PtdIns3P-binding WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that upon inhibition of mATG8 lipidation in cells, there is a decreased accumulation of WIPI2, suggesting a feedback loop between mATG8s and PtdIns3P production. The role of PtdIns3K-C1 in this feedback was demonstrated by in vitro experiments where recombinant membrane-coupled mATG8s bind to and potently activate PtdIns3K-C1, with GABARAP being the most potent activator among all mATG8s. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP binds two sites in PtdIns3K-C1, with one site showing an atypical bipartite interaction with the mATG8. We also confirm both sites are essential for GABARAP to activate PtdIns3K-C1. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be synthesized within a brief period. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/712327v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dc398eorg.highwire.dtl.DTLVardef@146a2cborg.highwire.dtl.DTLVardef@69c649org.highwire.dtl.DTLVardef@58fd81_HPS_FORMAT_FIGEXP M_FIG The GABARAP-PtdIns3K-C1 positive feedback loop. Model for the GABARAP-PtdIns3K-C1 positive feedback loop. GABARAP is indirectly recruited to the growing phagophore by PtdIns3P and activates PtdIns3K-C1, leading to an increased PtdIns3P production. The E1 (ATG7), E2 (ATG3) and E3 (ATG5-ATG12-ATG16L1) enzymes and WIPI2 are involved in the lipidation (covalent coupling) of GABARAP to membranes. C_FIG

molecular biology↗

bioRxiv2025

Structural and functional analysis of the Mycobacterium tuberculosis MmpS5L5 efflux pump presages a pathway to increased bedaquiline resistance

Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multi drug resistant tuberculosis. Clinical bedaquiline resistance in Mycobacterium tuberculosis has rapidly emerged, primarily due to mutations in the transcriptional repressor, Rv0678 that result in upregulation of the Resistance-Nodulation-Division (RND) efflux pump MmpS5/MmpL5 (MmpS5L5). Here, to understand how MmpS5L5 effluxes bedaquiline, we determined the structure of the MmpS5L5 complex using cryo-electron microscopy, revealing a novel trimeric architecture distinct from the canonical tripartite RND efflux pumps of Gram-negative bacteria. Structure prediction modelling in conjunction with functional genetic analysis indicates that it uses a periplasmic coiled-coil tube to transport molecules across the cell wall. Structure-guided genetic approaches identify MmpL5 mutations that alter bedaquiline transport; these mutations converge on a region in MmpL5 located in the lower portion of the periplasmic cavity, proximal to the outer leaflet of the inner membrane, suggesting a route for bedaquiline entry into the pump. While currently known clinical resistance to bedaquiline is due to pump upregulation, our findings that several MmpL5 variants increase bedaquiline efflux may presage the emergence of additional modes of clinical resistance. Significance StatementResistance to bedaquiline, a cornerstone drug for treating multidrug-resistant tuberculosis, is rapidly emerging due to mutations that upregulate expression of the MmpS5L5 efflux pump. Here, we reveal the cryo-EM structure of this pump, showing a novel trimeric architecture and a unique -helical coiled-coil tube for drug transport. Structure-guided genetic analysis identifies MmpL5 variants that further increase bedaquiline efflux, suggesting potential resistance mechanisms beyond pump upregulation.

microbiology↗

bioRxiv2025

Chimpanzee and human ApoE isoforms differ in the stimulation of neurite differentiation consistent with structural predictions with relevance to brain development and aging

BackgroundAmong anthropoids, humans uniquely possess ApoE isoforms that modulate Alzheimers disease (AD) risk and brain aging. While chimpanzee and human ApoE4 share R112 and R158, chimps do not exhibit advanced AD. A key difference is T61 in chimps versus R61 in humans, structurally resembling ApoE3. ObjectiveWe examined how astrocyte-derived ApoE isoforms impact neuronal morphology and used structural modeling to explore functional divergence. MethodsNeonatal rat hippocampal neurons were cultured with astrocyte-conditioned media (ACM) from mice expressing human ApoE3, ApoE4, or chimpanzee ApoE. Neuronal outgrowth was quantified after 72 hours. ResultsChimpanzee ACM increased neurite number by 30% over human ApoE isoforms. However, chimpanzee ACM resembled ApoE4 functionally, producing 40% shorter neurites and spines. Structural modeling supported greater similarity to ApoE4. ConclusionsChimpanzee ApoE is structurally and functionally more similar to ApoE4 than ApoE3, revealing evolutionary distinctions relevant to AD risk and neurodevelopment.

neuroscience↗

bioRxiv2024

The intracellular bacterium Orientia tsutsugamushi hijacks the adaptor protein BICD2 for dynein-based motility

The intracellular bacterium Orientia tsutsugamushi relies on the microtubule cytoskeleton and the motor protein dynein to traffic to the perinuclear region within infected cells. However, it remains unclear how the bacterium is coupled to the dynein machinery and how transport is regulated. Here, we discover that O. tsutsugamushi uses its autotransporter protein ScaC to recruit the dynein adaptor BICD2 to the bacterial surface. We show that ScaC is sufficient to engage dynein-based motility in the absence of other bacterial proteins and that BICD2 is required for efficient movement of O. tsutsugamushi during infection. Using TIRF single-molecule assays, we demonstrate that ScaC induces BICD2 to adopt an open conformation which activates the assembly of dynein-dynactin complexes. Our results reveal a novel role for BICD2 during bacterial infection and provide mechanistic insights into the life cycle of an important human pathogen.

microbiology↗

bioRxiv2023

KIF1C activates and extends dynein movement through the FHF cargo adaptor

Cellular cargos move bidirectionally on microtubules due to the presence of opposite polarity motors dynein and kinesin. Many studies show these motors are co-dependent, whereby one requires the activity of the other, although the mechanism is unknown. Here, using in vitro motility assays, we show that the kinesin-3 KIF1C acts both as an activator and a processivity factor for dynein. Activation only requires a fragment of the non-motor tail of KIF1C (KIF1C-stalk) to bind the cargo adaptor HOOK3. Cryo-EM, crosslinking mass spectrometry and AlphaFold2 predictions reveal this binding site to be separate from that of two constitutive factors (FTS and FHIP), which link HOOK3 to small G-proteins on cargos. We provide a structural model for how the FTS-HOOK3-FHIP1B (FHF) complex is auto-inhibited and explain how the KIF1C-stalk relieves this inhibition. Collectively, our work provides a molecular explanation for co-dependency by revealing that the mutual activation of dynein and kinesin is mediated through their shared adaptor. Many adaptors bind both dynein and kinesins, suggesting this mechanism could be generalised to other bidirectional complexes.

cell biology↗

bioRxiv2023

Alzheimer's Disease associations of ferritin and glutathione with oxidative damage and neuronal loss

ABSTRACTIron-mediated cell death (ferroptosis) is a proposed mechanism of Alzheimers disease (AD) pathology. While iron is essential for basic biological functions, its reactivity generates oxidants which contribute to cell damage and death. To further resolve mechanisms of iron-mediated toxicity in AD, we analyzed postmortem human brain and ApoEFAD mice. AD brains had decreased antioxidant enzymes, including those mediated by glutathione (GSH). Subcellular analyses of AD brains showed greater oxidative damage and lower antioxidant enzymes in lipid rafts, the site of amyloid processing, than in the non-raft membrane fraction. ApoE4 carriers had lower lipid raft yield with greater membrane oxidation. The hypothesized role of iron to AD pathology was tested in ApoEFAD mice by iron chelation with deferoxamine, which decreased fibrillar amyloid and lipid peroxidation, together with increased GSH-mediated antioxidants. These novel molecular pathways in iron mediated damage during AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/534324v3_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1a28ddborg.highwire.dtl.DTLVardef@10f57f7org.highwire.dtl.DTLVardef@583c8corg.highwire.dtl.DTLVardef@ef45f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Hypothesis: AD brain lipid peroxidation is driven by increased brain iron and decreased antioxidant defenses. Schema shows proteins that mediate iron metabolism in relation to lipid peroxidation (HNE) and antioxidant defenses in prefrontal cortex. AD-associated increase (red), decrease (blue), or no change (grey), relative to cognitively normal elderly controls. A{beta}; amyloid beta, ALDH2; alcohol dehydrogenase, APP; amyloid precursor protein, DMT1; divalent metal transporter 1; FPN, ferroportin; FSP1, ferroptosis suppressor protein 1, which requires the quinol cycle to attenuate lipid peroxidation; FTH1, ferritin heavy chain; FTL; ferritin light chain; GCLC, glutathione cysteine ligase catalytic subunit; GCLM, glutathione cysteine ligase modulator; GPx4, glutathione peroxidase 4; GSH, glutathione; GSSG, glutathione disulfide; GSTA4, glutathione S-transferase A4; HMOX; heme oxygenase; IRP, iron regulatory protein; LAT1, large neutral amino acid transporter 1; LOOH, Lipid hydroperoxides; Nrf2, Nuclear factor erythroid 2-related factor 2; Prdx6, peroxiredoxin 6; TF, transferrin, TfR; Transferrin receptor; xCT, cysteine-glutamate antiporter.

neuroscience↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2021

A native mass spectrometry-based assay for rapid assessment of the empty/full capsid ratio in AAV gene therapy products

Adeno-associated virus (AAV)-based cell and gene therapy is a rapidly developing field, requiring analytical methods for detailed product characterization. One important quality attribute of AAV products that requires monitoring is the amounts of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. Here, we present a native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation. We report rapid determination of the amount of empty particles in AAV5 and AAV8 samples, with results correlating well with more conventional analysis strategies, demonstrating the potential of state-of-the-art mass spectrometry for the characterization of viral particles.

biochemistry↗