Matthew Savoian - Academia.edu (original) (raw)

Papers by Matthew Savoian

Journal of Cell Science, 2005

Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which... more Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted...

Additional file 1: Figure S1. Sf9 cells were infected with the baculovirus expressing indicated h... more Additional file 1: Figure S1. Sf9 cells were infected with the baculovirus expressing indicated heterologous transgenes and cultured for 24, 36 or 48 h before processing. (A) Flag-ATM (red) is found in the nucleus (identified by DAPI staining; blue) as well as in the cytosplasm (cell boundaries are visible in the DIC image) at all-time points. (B) HA-p400 (green) is also distributed in all cellular compartments 24 h after infection. As time advances (36, 48 h), the protein exits from the nucleus (delineated by DAPI; blue) until it is exclusively cytoplasmic. (C) cells co-expressing Flag-ATM (red) and HA-p400 (green) exhibit dynamic protein relocalisation. 24 h post-infection a common pool of ATM and p400 is observed in the nucleus (DAPI; blue). The nuclear localisation of both proteins decreases with a concomitant enrichment of the cytoplasmic fraction (36, 48 h). All scale bars are 5 µm.

Nature Communications, 2015

Additional file 2: Figure S2. (A) ectopic expression of ATM-interacting p400 fragments in U2OS ce... more Additional file 2: Figure S2. (A) ectopic expression of ATM-interacting p400 fragments in U2OS cells. U2OS cells were infected with lentivirus expressing Flag-tagged p400 fragments and selected for 5 days against puromycin. Total 500 μg of protein was immunoprecipitated with M2 agarose and analysed by immunoblotting with anti-Flag antibody. (B) growth curve of puromycin-selected U2OS cells. U2OS cells were infected with lentivirus expressing Flag-F1or Flag-F6, and selected for 5 days against puromycin. Each cell line was seeded in 96-well plate in triplicate and monitored for the cell proliferation.

Nature cell biology, 2000

Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuat... more Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in ...

Although Drosophila larval neuroblasts are routinely used to define mutations affecting mitosis, ... more Although Drosophila larval neuroblasts are routinely used to define mutations affecting mitosis, the dynamics of karyokinesis in this system remain to be described. Here we outline a simple method for the short-term culturing of neuroblasts, from Drosophila third instar larvae, that allows mitosis to be followed by high-resolution multi-mode light microscopy. At 24 degrees C, spindle formation takes 7+/-0.5 minutes. Analysis of neuroblasts containing various GFP-tagged proteins (e.g. histone, fizzy, fizzy-related and alpha-tubulin) reveals that attaching kinetochores exhibit sudden, rapid pole-directed motions and that congressing and metaphase chromosomes do not undergo oscillations. By metaphase, the arms of longer chromosomes can be resolved as two chromatids, and they often extend towards a pole. Anaphase A and B occur concurrently, and during anaphase A chromatids move poleward at 3.2+/-0.1 microm/minute, whereas during anaphase B the spindle poles separate at 1.6+/-01 microm/m...

Molecular Plant-Microbe Interactions®

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic ... more Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall–remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption...

International Journal of Systematic and Evolutionary Microbiology

Six isolates of Campylobacter with similar non-standard colonial morphologies were identified dur... more Six isolates of Campylobacter with similar non-standard colonial morphologies were identified during studies isolating Campylobacter from bird faeces and rivers in New Zealand. Genomic (16S rRNA gene sequencing and whole genome analysis) and phenotypic (MALDI-TOF analysis and conventional biochemical tests) showed that the isolates form a monophyletic clade with genetic relationships to Campylobacter coli / Campylobacter jejuni and Campylobacter peloridis /Campylobacter amoricus. They may be distinguished from other Campylobacter by their MALDI-TOF spectral pattern, their florid α-haemolysis, their ability to grow anaerobically at 37 °C, and on 2 % NaCl nutrient agar, and their lack of hippuricase. This study shows that these isolates represent a novel species within the genus Campylobacter for which the name Campylobacter novaezeelandiae sp. nov. is proposed. The presence of C. novaezeelandiae in water may be a confounder for freshwater microbial risk assessment as they may not be ...

Neuroblast (NB) cell division is characterized by a basal positioning of the cleavage furrow resu... more Neuroblast (NB) cell division is characterized by a basal positioning of the cleavage furrow resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly is governed by centralspindlin, a highly conserved complex that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In contrast to model systems studied so far, these two centralspindlin populations are spatially and temporally separated in NBs. A cortical leading pool is located at the basal cleavage furrow site and a second pool accumulates at the midzone before travelling to the site of the basal cleavage furrow during cytokinesis completion. By manipulating microtubule (MT) dynamics, we show that the cortical centralspindlin population requires peripheral astral microtubules and the Chromosome Passenger Complex (CPC) for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin leve...

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is k... more Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyse the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analysed by light-, confocal-and electron microscopy. Changes in PA distribution were analysed in E. festucae using a PA biosensor and the impact of these changes on endocytic recycling and superoxide production investigated. We found that E. festucae PldB and the N. crassa ortholog, PLA-7, are required for polarized growth, cell fusion and ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network that controls hyphal morphogenesis and growth in filamentous fungi.

Molecular Plant Pathology

Microbial Genomics

Campylobacter jejuni is the most common cause of bacterial diarrheal disease in the world. Clinic... more Campylobacter jejuni is the most common cause of bacterial diarrheal disease in the world. Clinical outcomes of infection can range from asymptomatic infection to life-threatening extraintestinal infections. This variability in outcomes for infected patients has raised questions as to whether genetic differences between C. jejuni isolates contribute to their likelihood of causing severe disease. In this study, we compare the genomes of ten C. jejuni isolates that were implicated in extraintestinal infections with reference gastrointestinal isolates, in order to identify unusual patterns of sequence variation associated with infection outcome. We identified a collection of genes that display a higher burden of uncommon mutations in invasive isolates compared with gastrointestinal close relatives, including some that have been previously linked to virulence and invasiveness in C. jejuni. Among the top genes identified were mreB and pgp1, which are both involved in determining cell shape. Electron microscopy confirmed morphological differences in isolates carrying unusual sequence variants of these genes, indicating a possible relationship between extraintestinal infection and changes in cell morphology. DATA SUMMARY (1) Sequence data has been deposited in the Sequence Read Archive; accession number PRJNA475221 (URL-https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA475221). (2) Lists of orthologous genes, corresponding bitscore data and code used for this analysis have been deposited in GitHub (URL-https://github.com/Gardner-BinfLab/invasive_campylobacter).

PloS one, 2017

This is the first integrated study of the effects on gastric secretion, inflammation and fundic m... more This is the first integrated study of the effects on gastric secretion, inflammation and fundic mucins after infection with L3 T. circumcincta and in the very early period following transplantation of adult worms. At 3 months-of-age, 20 Coopworth lambs were infected intraruminally with 35,000 L3; infected animals were killed on Days 5, 10, 15, 20 and 30 post-infection and 6 controls on either Day 0 or 30 post-infection. Another 15 Romney cross lambs received 10,000 adult worms at 4-5 months-of-age though surgically-implanted abomasal cannulae and were killed after 6, 12, 24 and 72 hours; uninfected controls were also killed at 72 hours. Blood was collected at regular intervals from all animals for measurement of serum gastrin and pepsinogen and abomasal fluid for pH measurement from cannulated sheep. Tissues collected at necropsy were fixed in Bouin's fluid for light microscopy, immunocytochemistry and mucin staining and in Karnovsky's fluid for electron microscopy. Nodules ...

Methods in Molecular Biology, 2017

The spindle is a microtubule-based structure whose remodeling is required for partitioning the ch... more The spindle is a microtubule-based structure whose remodeling is required for partitioning the chromosomes and cytoplasm during meiosis. Characterizing microtubule behavior is fundamental to understanding how these tubulin polymers contribute to successful cell division. Here, a procedure is described for the imaging and analysis of spindle microtubule dynamics in cultures of living Drosophila melanogaster primary spermatocytes expressing tubulin tagged with enhanced green fluorescent protein. It employs time-lapse scanning confocal microscopy and the photobleaching of fiduciary marks onto fluorescently tagged microtubules. These labels are subsequently used to determine the sites and rates of kinetochore fiber growth and shrinkage during metaphase. This method can be readily applied to different microtubule populations, meiotic stages, and genetic backgrounds.

BMC molecular biology, Nov 4, 2016

Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinas... more Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Het...

Journal of biomolecular techniques : JBT, Jan 19, 2015

In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. C... more In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during differ...

Open biology, 2014

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and s... more Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while i...

eLS, 2001

The fruitfly Drosophila melanogaster offers a rich and varied source of differentiated cell types... more The fruitfly Drosophila melanogaster offers a rich and varied source of differentiated cell types. This and its exceptional experimental tractability make it well suited for cell cycle or other developmental biology investigations. In particular, the sperm-producing cells of the testes provide a powerful system for studying the mechanics of cell division. These meiotically dividing primary spermatocytes can be easily isolated from the testes of mutant or transgenic animals and maintained in short-term primary cultures. The cells' large and flat geometry makes them amenable to a variety of live cell light-microscopy-based observation methods. Single-plane transmitted light time-lapse imaging and more advanced multi-dimensional widefield or confocal fluorescence microscopy have been used to define the morphological and kinetic changes that accompany processes ranging from meiotic chromosome segregation to microtubule dynamics. The ability to document such dynamic changes underscores the power of live cell imaging in understanding cell physiology and function. Key Concepts Drosophila is an experimentally tractable source of different cell types. Male meiotic primary spermatocytes provide a powerful model system for studying cell division. These large flat cells are easily cultured for live cell light microscopy. Transmitted light and fluorescence imaging methods can be used separately or in tandem to record different dynamic processes during cell division. Keywords: live cell imaging; mitosis; meiosis; light microscopy; fluorescent protein