Visual inspection of histopathology slides is one of the main methods used by pathologists to assess the stage, type and subtype of lung tumors. Adenocarcinoma (LUAD) and squamous cell carcinoma ...(LUSC) are the most prevalent subtypes of lung cancer, and their distinction requires visual inspection by an experienced pathologist. In this study, we trained a deep convolutional neural network (inception v3) on whole-slide images obtained from The Cancer Genome Atlas to accurately and automatically classify them into LUAD, LUSC or normal lung tissue. The performance of our method is comparable to that of pathologists, with an average area under the curve (AUC) of 0.97. Our model was validated on independent datasets of frozen tissues, formalin-fixed paraffin-embedded tissues and biopsies. Furthermore, we trained the network to predict the ten most commonly mutated genes in LUAD. We found that six of them-STK11, EGFR, FAT1, SETBP1, KRAS and TP53-can be predicted from pathology images, with AUCs from 0.733 to 0.856 as measured on a held-out population. These findings suggest that deep-learning models can assist pathologists in the detection of cancer subtype or gene mutations. Our approach can be applied to any cancer type, and the code is available at https://github.com/ncoudray/DeepPATH .
Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial ...spoke (RS) protein complexes and the microtubule central pair (CP). Despite their importance for ciliary motility across eukaryotes, the molecular function of the RSs is unknown. Here, we reconstituted the Chlamydomonas reinhardtii RS head that abuts the CP and determined its structure using single-particle cryo-EM to 3.1-Å resolution, revealing a flat, negatively charged surface supported by a rigid core of tightly intertwined proteins. Mutations in this core, corresponding to those involved in human ciliopathies, compromised the stability of the recombinant complex, providing a molecular basis for disease. Partially reversing the negative charge on the RS surface impaired motility in C. reinhardtii. We propose that the RS-head architecture is well-suited for mechanoregulation of ciliary beating through physical collisions with the CP.
Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely ...how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified
γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.
YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of ...an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.
ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal ...structure of MlaFB from
, the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family.
The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer‐binding, and mechanical elements. Such communication is particularly complex and ...not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule‐binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled‐coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule‐independent hyperactive ATPase activity, and nucleotide‐independent low affinity for microtubules. Cryo‐electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide‐dependent conformational changes occur normally in one‐half of the AAA ring, but are disrupted in the other half. The large‐scale linker conformational change observed in the wild‐type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.
Synopsis
The movement of dynein on microtubules requires communication between enzymatic, polymer‐binding, and mechanical elements. Our results reveal how dynein's coiled‐coil stalk plays a critical role in coordinating such domain movements.
Stalk mutants disrupt unidirectional motion along microtubules, show nucleotide‐independent low affinity for microtubules, and lack microtubule‐regulation of ATPase activity.
Cryo‐electron microscopy structures of one mutant show that nucleotide‐dependent conformational changes are disrupted in one half of the AAA ring while the other half is minimally affected; the partial ring conformational change results in unregulated ATP hydrolysis and blocks the linker conformational change that drives motility.
Our structural and functional results suggest a model for how the stalk domain modulates conformational changes around the AAA ring, which are initiated by nucleotide binding at dynein's main ATP site (AAA1).
Mutations in the dynein stalk demonstrate its role in coordinating conformational changes across the AAA ring of the motor domain, and in regulating microtubule‐dependent movement and ATPase activity.
In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE ...transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.
Abstract Cancer diagnosis and management depend upon the extraction of complex information from microscopy images by pathologists, which requires time-consuming expert interpretation prone to human ...bias. Supervised deep learning approaches have proven powerful, but are inherently limited by the cost and quality of annotations used for training. Therefore, we present Histomorphological Phenotype Learning, a self-supervised methodology requiring no labels and operating via the automatic discovery of discriminatory features in image tiles. Tiles are grouped into morphologically similar clusters which constitute an atlas of histomorphological phenotypes (HP-Atlas), revealing trajectories from benign to malignant tissue via inflammatory and reactive phenotypes. These clusters have distinct features which can be identified using orthogonal methods, linking histologic, molecular and clinical phenotypes. Applied to lung cancer, we show that they align closely with patient survival, with histopathologically recognised tumor types and growth patterns, and with transcriptomic measures of immunophenotype. These properties are maintained in a multi-cancer study.
YiiP is a dimeric Zn ²⁺/H ⁺ antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP ...homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn ²⁺. Starting from the X-ray structure in the presence of Zn ²⁺, we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.
Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate ...uptake in plants. The SLC4 family is more distantly related to members of the Amino acid‐Polyamine‐organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo‐electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo‐EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all‐atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward‐facing conformation. Comparisons of the resulting Bor1p model with the X‐ray structure of AE1 in an outward‐facing conformation, together with MD simulations of inward‐facing and outward‐facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking‐bundle transport mechanism described for other members of the APC superfamily.
PDB Code(s): 5SV9; EMD-8313
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