ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

Clathrin's adaptor interaction sites are repurposed to stabilize microtubules during mitosis.

Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.

Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

CDK-regulated dimerization of M18BP1 on a Mis18 hexamer is necessary for CENP-A loading.

Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18ß subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18ß 4:2 hexamer binds two copies of M18BP1 through M18BP1's 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18ß, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18ß scaffold.

GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK.

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.

Type II secretion: the substrates that won't go away.

Type II secretion systems (T2SSs) generally release their substrates into the culture medium. A few T2SS substrates remain anchored to or bound at the surface of the bacteria after secretion. Since they handle already folded proteins, T2SSs are the best way for bacteria to target, at their surface, proteins containing a cofactor, proteins that have to be folded in the cytoplasm or in the periplasm, or multimeric proteins. However, how a T2SS deals with membrane-anchored proteins is not yet understood. While this type of protein has until now been overlooked, new proteomic approaches will facilitate its identification.

SurA is involved in the targeting to the outer membrane of a Tat signal sequence-anchored protein.

The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii, the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of ß-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli, periplasmic chaperones SurA, Skp, and DegP work together with the ß-barrel assembly machinery (Bam) to target and insert ß-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system.

Comparing the substrate specificities of cytochrome c biogenesis Systems I and II: bioenergetics.

c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attachment of heme to the cytochrome polypeptide. Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subunits, two of which (ResB and ResC) are the minimal functional unit. The ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC. Heterologous expression of ResBC in Escherichia coli lacking its own biogenesis machinery allowed us to investigate the substrate specificity of System II. ResBC is able to attach heme to monoheme c-type cytochromes c(550) from Paracoccus denitrificans and c(552) from Hydrogenobacter thermophilus, both normally matured by System I. The production of holocytochrome is enhanced by the addition of exogenous reductant. Single-cysteine variants of these cytochromes were not efficiently matured by System II, but System I was able to produce detectable amounts of AXXCH variants; this adds to evidence that there is no obligate requirement for a disulfide-bonded intermediate for the latter c-type cytochrome biogenesis system. In addition, System II was able to mature an AXXAH-containing variant into a b-type cytochrome, with implications for both heme supply to the periplasm and substrate recognition by System II.