dictyNews
Electronic Edition
Volume 45, number 1
January 11, 2019
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Abstracts
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Contractility kits promote assembly of the mechanoresponsive
cytoskeletal network
Priyanka Kothari, Vasudha Srivastava, Vasudha Aggarwal, Irina
Tchernyshyov, Jennifer E. Van Eyk, Taekjip Ha, and Douglas N. Robinson
Journal of Cell Science, In press
Cellular contractility is governed by a control system of proteins that
integrates internal and external cues to drive diverse shape change processes.
This contractility controller includes myosin II motors, actin crosslinkers, and
protein scaffolds, which exhibit robust and cooperative mechanoaccumulation.
However, the biochemical interactions and feedback mechanisms that drive
the controller remain unknown. Here, we use a proteomics approach to identify
direct interactors of two key nodes of the contractility controller in the social
amoeba Dictyostelium discoideum: the actin crosslinker cortexillin I and the
scaffolding protein IQGAP2. We highlight several unexpected proteins that
suggest feedback from metabolic and RNA-binding proteins on the contractility
controller. Quantitative in vivo biochemical measurements reveal direct
interactions between myosin II and cortexillin I, which form the core
mechanosensor. Further, IQGAP1 negatively regulates mechanoresponsiveness
by competing with IQGAP2 for binding the myosin II-cortexillin I complex.
These myosin II-cortexillin I-IQGAP2 complexes are pre-assembled into higher
order mechanoresponsive contractility kits (MCKs) poised to integrate into the
cortex upon diffusional encounter coincident with mechanical inputs.
submitted by: Doug Robison [[log in to unmask]]
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Myosin IIB assembly state determines its mechanosensitive dynamics
Eric S. Schiffhauer, Yixin Ren, Vicente A. Iglesias, Priyanka Kothari,
Pablo A. Iglesias, Douglas N. Robinson
J. Cell Biol. In press
Dynamical cell shape changes require a highly sensitive cellular system that
can respond to chemical and mechanical inputs. Myosin IIs are key players
in the cell’s ability to react to mechanical inputs, demonstrating an ability to
accumulate in response to applied stress. Here we show that inputs that
influence the ability of myosin II to assemble into filaments impact the ability
of myosin to respond to stress in a predictable manner. Using mathematical
modeling for Dictyostelium myosin II, we predict that myosin II
mechanoresponsiveness will be biphasic with an optimum established by the
percentage of myosin II assembled into bipolar filaments. In HeLa and NIH 3T3
cells, heavy chain phosphorylation of NMIIB by PKC-Zeta, as well as
expression of NMIIA, can control the ability of NMIIB to mechanorespond by
influencing its assembly state. These data demonstrate that multiple inputs to
the myosin II assembly state integrate at the level of myosin II to govern the
cellular response to mechanical inputs.
submitted by: Doug Robison [[log in to unmask]]
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The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent
damage at the Mycobacterium-containing vacuole but have opposite impact on
containing the infection
Ana T. López-Jiménez1, Elena Cardenal-Muñoz1, Florence Leuba1, Lilli
Gerstenmaier2, Caroline Barisch1, Monica Hagedorn23, Jason S. King4 and
Thierry Soldati1
1Department of Biochemistry, Faculty of Science, University of Geneva,
Sciences II, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
2Section Parasitology, Bernhard Nocht Institute for Tropical Medicine,
20359 Hamburg, Germany.
3Life Sciences and Chemistry, Jacobs University Bremen gGmbH, group
Ribogenetics, Campus Ring 1, 28759 Bremen, Germany.
4Department of Biomedical Science, University of Sheffield, Western Bank,
Sheffield S10 2TN, United Kingdom.
PLoS Pathog 14(12): e1007501. https://doi.org/10.1371/journal.ppat.1007501
Phagocytic cells capture and kill most invader microbes within the bactericidal
phagosome, but some pathogens subvert killing by damaging the compartment
and escaping to the cytosol. To prevent the leakage of pathogen virulence and
host defence factors, as well as bacteria escape, host cells have to contain and
repair the membrane damage, or finally eliminate the cytosolic bacteria. All
eukaryotic cells engage various repair mechanisms to ensure plasma membrane
integrity and proper compartmentalization of organelles, including the Endosomal
Sorting Complex Required for Transport (ESCRT) and autophagy machineries.
We show that during infection of Dictyostelium discoideum with Mycobacterium
marinum, the ESCRT-I component Tsg101, the ESCRT-III protein
Snf7/Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to sites of damage
at the Mycobacterium-containing vacuole. Interestingly, damage separately
recruits the ESCRT and the autophagy machineries. In addition, the recruitment
of Vps32 and Vps4 to repair sterile membrane damage depends on Tsg101 but
appears independent of Ca2+. Finally, in absence of Tsg101, M. marinum
accesses prematurely the cytosol, where the autophagy machinery restricts its
growth. We propose that ESCRT has an evolutionary conserved function to
repair small membrane damage and to contain intracellular pathogens in intact
compartments.
submitted by: Thierry Soldati [[log in to unmask]]
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IQGAP-related protein IqgC suppresses Ras signaling during large-scale
endocytosis
Maja Marinović, Lucija Mijanović;, Marko Šoštar, Matej Vizovišek, Alexander
Junemann, Marko Fonović, Boris Turk, Igor Weber, Jan Faix and Vedrana Filić*
* Correspondence: [log in to unmask]
PNAS https://doi.org/10.1073/pnas.1810268116
Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk
endocytosis used by cells to ingest large volumes of fluid and solid particles,
respectively. Both processes are regulated by Ras signaling, which is precisely
controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs)
responsible for terminating Ras activity on early endosomes. While regulation of
Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for
the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in
commonly used axenic laboratory strains, this gene is mutated and inactive.
Moreover, none of the RasGAPs characterized so far have been implicated in the
regulation of Ras signaling in large-scale endocytosis in axenic strains. In this
study, we establish, using biochemical approaches and complementation assays
in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active
RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC- and
IqgC-overexpressing cells further revealed participation of this GAP in the
regulation of both types of large-scale endocytosis and in cytokinesis. Moreover,
given the localization of IqgC to phagosomes and, most prominently, to
macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale
endocytosis. The data presented here functionally distinguish IqgC from other
members of the Dictyostelium IQGAP family and call for repositioning of this
genuine RasGAP outside of the IQGAP group.
submitted by: Maja Marinović [[log in to unmask]]
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Symbiont location, host fitness, and possible coadaptation in a symbiosis between
social amoebae and bacteria.
Longfei Shu, Debra A. Brock, Katherine S. Geist, Jacob W. Miller, David C. Queller,
Joan E. Strassmann, Susanne DiSalvo.
Department of Biology, Washington University in St. Louis
eLife in press eLife 2018;7:e42660 doi: 10.7554/eLife.42660
Recent symbioses, particularly facultative ones, are well suited for unravelling the
evolutionary give and take between partners. Here we look at variation in natural
isolates of the social amoeba Dictyostelium discoideum and their relationships
with bacterial symbionts, Burkholderia hayleyella and Burkholderia agricolaris.
Only about a third of field-collected amoebae carry a symbiont. We cured and
cross-infected amoebae hosts with different symbiont association histories and
then compared host responses to each symbiont type. Before curing, field-collected
clones did not vary significantly in overall fitness, but infected hosts produced
morphologically different multicellular structures. After curing and reinfecting, host
fitness declined. However, natural B. hayleyella hosts suffered fewer fitness costs
when reinfected with B. hayleyella, indicating that they have evolved mechanisms
to tolerate their symbiont. Our work suggests that amoebae hosts have evolved
mechanisms to tolerate specific acquired symbionts; exploring host-symbiont
relationships that vary within species may provide further insights into disease
dynamics.
submitted by: Joan Strassmann [[log in to unmask]]
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The specificity of Burkholderia symbionts in the social amoeba farming symbiosis:
prevalence, species, genetic and phenotypic diversity.
Department of Biology, Washington Univrsity in St. Louis
Tamara S. Haselkorn, Susanne DiSalvo, Jacob W. Miller, Usman Bashir,
Debra A. Brock, David C. Queller, and Joan E. Strassmann.
Molecular Ecology in press https://doi.org/10.1111/mec.14982.
The establishment of symbioses between eukaryotic hosts and bacterial symbionts
in nature is a dynamic process. The formation of such relationships depends on the
life history of both partners. Bacterial symbionts of amoebae may have unique
evolutionary trajectories to the symbiont lifestyle, because bacteria are typically
ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis.
In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia
bacteria are able to resist amoebal digestion and maintain a persistent relationship
that includes carriage throughout the amoeba's social cycle that culminates in spore
formation. Some Burkholderia strains allow their host to carry other bacteria, as food.
This carried food is released in new environments in a trait called farming. To better
understand the diversity and prevalence of Burkholderia symbionts and the traits
they impart to their amoebae hosts, we first screened 700 natural isolates of
D. discoideum and found 25% infected with Burkholderia. We next used a multilocus
phylogenetic analysis and identified two independent transitions by Burkholderia to
the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from
D. discoideum, as well as strains isolated from other sources, for traits relevant to
symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the
B. agricolaris, B. hayleyella, and B. bonniea species were able to form persistent
symbiotic associations with D. discoideum. The Burkholderia‐Dictyostelium
relationship provides a promising arena for further studies of the pathway to
symbiosis in a unique system.
submitted by: Joan Strassmann [[log in to unmask]]
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Burkholderia bacteria use chemotaxis to find social amoeba Dictyostelium discoideum
hosts.
Department of Biology, Washington Univrsity in St. Louis
Longfei Shu, Bojie Zhang, David C. Queller, and Joan E. Strassmann, J.E. 2018.
ISME Journal 12:1977–1993https://www.nature.com/articles/s41396-018-0147-4
A key question in cooperation is how to find the right partners and maintain cooperative
relationships. This is especially challenging for horizontally transferred bacterial
symbionts where relationships must be repeatedly established anew. In the social
amoeba Dictyostelium discoideum farming symbiosis, two species of inedible
Burkholderia bacteria (Burkholderia agricolaris and Burkholderia hayleyella) initiate
stable associations with naive D. discoideum hosts and cause carriage of additional
bacterial species. However, it is not clear how the association between D. discoideum
and its carried Burkholderia is formed and maintained. Here, we look at precisely how
Burkholderia finds its hosts. We found that both species of Burkholderia clones isolated
from D. discoideum, but not other tested Burkholderia species, are attracted to
D. discoideum supernatant, showing that the association is not simply the result of
haphazard engulfment by the amoebas. The chemotactic responses are affected by
both partners. We find evidence that B. hayleyella prefers D. discoideum clones that
currently or previously carried Burkholderia, while B. agricolaris does not show this
preference. However, we find no evidence of Burkholderia preference for their own host
clone or for other hosts of their own species. We further investigate the chemical
differences of D. discoideum supernatants that might explain the patterns shown above
using a mass spectrometry based metabolomics approach. These results show that
these bacterial symbionts are able to preferentially find and to some extent choose their
unicellular partners. In addition, this study also suggests that bacteria can actively
search for and target phagocytic cells, which may help us better understand how
bacteria interact with immune systems.
submitted by: Joan Strassmann [[log in to unmask]]
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B447, RB448, RB449, RB450, RB451 and RB453 antibodies recognize a
Dictyostelium AlyL protein by Western blot.
Otmane Lamrabet, Tania Jauslin
Antibody Reports, 2018, 1:e02.
https://doi.org/10.24450/journals/abrep.2018.e2
The recombinant antibodies RB447, RB448, RB449, RB450, RB451 and RB453
detect by western blot the full-length AlyL protein from Dictyostelium discoideum.
submitted by: Wanessa du Fresne von Hohenesche [[log in to unmask]]
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RB285, RB286, RB287, RB288, RB289 and RB290 antibodies recognize a
Dictyostelium NcfA peptide by ELISA
Philippe Hammel, Wanessa Cristina Lima
Antibody Reports, 2018, 1:e04.
https://doi.org/10.24450/journals/abrep.2018.e4
The recombinant antibodies RB285, RB286, RB287, RB288, RB289 and RB290
detect by ELISA a synthetic peptide from the Dictyostelium NcfA protein.
submitted by: Wanessa du Fresne von Hohenesche [[log in to unmask]]
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