The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Maternal transfer of strain-specific immunity in an invertebrate

The most celebrated component of the vertebrate immune system is the acquired response in which memory cells established during primary infection enhance the proliferation of antibodies during secondary infection. Additionally, the strength of vertebrate acquired immune responses varies dramatically depending on the infecting pathogen species or on the pathogen genotype within species. Because invertebrates lack the T-cell receptors and Major Histocompatibility Complex (MHC) molecules that mediate vertebrate adaptive immune responses, they are thought to lack adaptive immunity and be relatively unspecific in their interactions with pathogens. With only innate immunity, invertebrate hosts are believed to be nai;ve at each new encounter with pathogens. Nevertheless, some forms of facultative immunity appear to be important in insects; some individuals have enhanced immunity due to population density, and some social insects benefit when their nest-mates have been exposed to a pathogen or pathogen mimic (; see for a predation example.) Here we provide evidence for acquired strain-specific immunity in the crustacean Daphnia magna infected with the pathogenic bacteria Pasteuria ramosa. Specifically, the fitness of hosts was enhanced when challenged with a bacterial strain their mother had experienced relative to cases when mother and offspring were challenged with different strains.     

A 96-well DNase I footprinting screen for drug–DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.     

Report on the Polycystididae (Rhabdocoela, Kalyptorhynchia) from Australia, with the Description of Twelve New Species and Six New Genera

Twelve new species of Polycystididae Graff, 1905 are described from the Australian east coast. Nine of them could not be placed in any of the existing genera, and therefore six new genera are erected. Alchoides n. gen. differs from all other polycystidid genera by the presence of a bundle of glands that opens into the distal part of the male atrium (accessory vesicle type V). Two species are included in the genus, A. alchoides n. sp. and A. dittmannae n. sp., which differ from each other in the shape of the stylet. Ametochus gehrkei n. gen n. sp. can be distinguished from other polycystidids by the presence of a prostate vesicle type III associated with a prostate stylet type III, combined with the presence of an accessory stylet type III, while a prostate stylet type II is lacking. Typical for Arrawarria inexpectata n. gen n. sp. is the combined presence of an armed cirrus with a prostate vesicle type II connected to a prostate stylet type II. Two species are assigned to the new genus Duplexostylus n. gen.: D. rowei n. sp. and D. winsori n. sp. These two species have two unique features: a prostate vesicle type IV directly connected to a prostate stylet type III and an asymmetrical septum that surrounds the proximal part of the male atrium. They can be distinguished from each other by differences in the detailed construction of their stylets. Two new species are placed in the new genus Stradorhynchus n. gen.: S. caecus n. sp. and S. terminalis n. sp. They have unpaired gonads and a prostate vesicle type IV connected to a prostate stylet type IV, which combination of characters makes them unique within the Polycystididae. The two species differ from each other in the shape and size of the stylet. Triaustrorhynchus armatus n. gen., n. sp. is the first species of the Polycystididae to have three types of stylet in the male atrium: a prostate stylet type II, a prostate stylet type III and an accessory stylet type III. Three new species could be placed in existing genera: Cincturorhynchus monaculeus n. sp., Paraustrorhynchus caligatus n. sp. and Polycystis australis n. sp. All three differ from their congeners in the form and dimensions of their stylets. All newly described species are discussed, giving their similarities and differences with resembling species. Austrorhynchus hawaiiensis Karling, 1977 is reported for the first time in Australia, and the Australian population is compared with populations from other regions. The presence of Gyratrix hermaphroditus Ehrenberg, 1831, one new species of Paulodora Marcus, 1948 and a new genus of Typhlopolycystidinae Evdonin, 1977 is mentioned.     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.