The Arp2/3 complex generates branched actin networks at different locations of the cell. The WASH and WAVE Nucleation Promoting Factors (NPFs) activate the Arp2/3 complex at the surface of endosomes ...or at the cell cortex, respectively. In this review, we will discuss how these two NPFs are controlled within distinct, yet related, multiprotein complexes. These complexes are not spontaneously assembled around WASH and WAVE, but require cellular assembly factors. The centrosome, which nucleates microtubules and branched actin, appears to be a privileged site for WASH complex assembly. The actin and microtubule cytoskeletons are both responsible for endosome shape and membrane remodeling. Motors, such as dynein, pull endosomes and extend membrane tubules along microtubule tracks, whereas branched actin pushes onto the endosomal membrane. It was recently uncovered that WASH assembles a super complex with dynactin, the major dynein activator, where the Capping Protein (CP) is exchanged from dynactin to the WASH complex. This CP swap initiates the first actin filament that primes the autocatalytic nucleation of branched actin at the surface of endosomes. Possible coordination between pushing and pulling forces in the remodeling of endosomal membranes is discussed.
Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in
and
tumor suppressor genes (TSGs). We generated single and combined
and
knock-outs (KOs) in the ...immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of
, but not
, induced the formation of large colonies in soft agar.
inactivation in
KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon
and
KO. Inactivation of
enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast,
knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.
During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 ...inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid-liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the
locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.
The diversity of prokaryotic symbionts in Ciliophora and other protists is fascinatingly rich; they may even include some potentially pathogenic bacteria. In this review, we summarize currently ...available data on biodiversity and some morphological and biological peculiarities of prokaryotic symbionts mainly within the genera Paramecium and Euplotes. Another direction of ciliate symbiology, neglected for a long time and now re‐discovered, is the study of epibionts of ciliates. This promises a variety of interesting outcomes. Last, but not least, we stress the new technologies, such as next generation sequencing and the use of genomics data, which all can clarify many new aspects of relevance. For this reason, a brief overview of achievements in genomic studies on ciliate's symbionts is provided. Summing up the results of numerous scientific contributions, we systematically update current knowledge and outline the prospects as to how symbiology of Ciliophora may develop in the near future.
spp. and "
Gortzia infectiva", known as
-like bacteria (HLB), are commonly found as nuclear endosymbionts of ciliates, especially the
genus. HLB are related by phylogenetic relationships, ...morphological features, and life-cycles, which involve two alternating morphotypes: reproductive and infectious forms (RF, IF). In this paper we describe a novel species belonging to the "
. Gortzia" genus, detected in
, a ciliate for which infection by an HLB has not been reported, discovered in India. This novel endosymbiont shows unusual and surprising features with respect to other HLB, such as large variations in IF morphology and the occasional ability to reproduce in the host cytoplasm. We propose the name of "
Gortzia shahrazadis" for this novel HLB. Moreover, we report two additional species of HLB from Indian
populations: "
. Gortzia infectiva" (from
), and
(from
); the latter is the first record of
from a tropical country. Although tropical, we retrieved
at an elevation of 706 m corresponding to a moderate climate not unlike conditions where
are normally found, suggesting the genus
does exist in tropical countries, but restricted to higher elevations.
Epithelial–mesenchymal transition (EMT) is a critical step in tumor progression that leads to the acquisition by cancer cells the capacity for migration using the mesenchymal motility mode regulated ...by the Rac→WAVE→Arp2/3 signaling pathway. Earlier it was shown that proteins interacting with Rac can regulate mesenchymal migration and thus determine the metastatic potential of the cells. The search for new regulators of cell migration is an important theoretical and practical task. The adaptor protein Anks1a is one of the proteins interacting with Rac, whose expression is altered in many types of tumors. The aim of this study was to find whether Anks1a affects the migration of cancer cells and to identify the mechanism underlying this effect. It was suggested that Anks1a can influence cancer cell migration either as a Rac1 effector or by activating human epidermal growth factor receptor 2 (HER2) exchange. We investigated how upregulation and inhibition of Anks1a expression affected migration of breast cancer cells with different HER2 status. Anks1a was shown to interact with the activated form of Rac1. In the MDA-MB-231 cells (triple negative cancer), which lack HER2, Anks1a accumulated at the active cell edge, which is characterized by enrichment with active Rac1, whereas no such accumulation was observed in the HER2-overexpressing SK-BR-3 cells. Downregulation of the
ANKS1a
expression with esiRNA had almost no effect on the cancer cell motility, except a slight increase in the average migration rate of MDA-MB-231 cells. Among three cell lines tested, overexpression of Anks1a increased the migration rate of HER2-overexpressng SK-BR-3 cells only. We showed that Anks1a is an effector of activated Rac1, but its influence on the cell migration in this capacity was minimal, at least in the studied breast cancer cells. Anks1a affected the motility of breast cancer cells due to its involvement in the EGF receptor exchange.
Branched actin networks polymerized by the Actin-related protein 2 and 3 (Arp2/3) complex play key roles in force generation and membrane remodeling. These networks are particularly important for ...cell migration, where they drive membrane protrusions of lamellipodia. Several Arp2/3 inhibitory compounds have been identified. Among them, the most widely used is CK-666 (2-Fluoro-N-2-(2-methyl-1H-indol-3-yl)ethyl-benzamide), whose mode of action is to prevent Arp2/3 from reaching its active conformation. Here 74 compounds structurally related to CK-666 were screened using a variety of assays. The primary screen involved EdU (5-ethynyl-2'-deoxyuridine) incorporation in untransformed MCF10A cells. The resulting nine positive hits were all blocking lamellipodial protrusions and cell migration in B16-F1 melanoma cells in secondary screens, showing that cell cycle progression can be a useful read-out of Arp2/3 activity. Selected compounds were also characterized on sea urchin embryos, where Arp2/3 inhibition yields specific phenotypes such as the lack of triradiate spicules and inhibition of archenteron elongation. Several compounds were filtered out due to their toxicity in cell cultures or on sea urchin development. Two CK-666 analogs,
(N-{2-5-(Benzyloxy)-2-methyl-1H-indol-3-yl ethyl}-3-bromobenzamide) and
(2,4-Dichloro-N-2-(7-chloro-2-methyl-1H-indol-3-yl) ethyl-5-(dimethylamino) sulfonyl benzamide), were active in all assays and significantly more efficient
than CK-666. These best hits with increased
potency were, however, slightly less efficient
than CK-666 in the classical pyrene-actin assay. Induced-fit docking of selected compounds and their possible metabolites revealed interaction with Arp2/3 that suppresses Arp2/3 activation. The data obtained in our screening validated the applicability of original assays for Arp2/3 activity. Several previously unexplored CK-666 structural analogs were found to suppress Arp2/3 activation, and two of them were identified as Arp2/3 inhibitors with improved
efficiency.
Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to ...agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus
(
,
) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the
-Like Organism (RLO) endosymbiont "
(
) Trichorickettsia mobilis" was identified in the macronucleus of the ciliate
. We performed transfection experiments of this RLO to planarians (
) per
. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians' enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by
or other RLOs and occurring in fish farms or in the wild.
Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the ...non‐coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free‐living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.
This article touches upon the issue of determination of external force factors influencing an unmanned aerial vehicle or drone in critical flight modes for further transition to the full-scale ...engineering development. A brief review of the design of the developing vehicle is given. A simplified three-dimensional model is created. On the basis of the 3d model and with the help of the FloEFD computer complex, the aerodynamic analysis of the chosen design of the developed unmanned aerial vehicle is carried out. The dependences of the available speed and disposable tangential overloads of flight acceleration are obtained. The minimum permissible horizontal flight acceleration is calculated. The aerodynamic coefficients of the used wing profile are given. The value of the available tangential overload for different flight speeds is defined. The relation of the maximum trajectory angle of inclination against the flight speed when flying above the ground is calculated. The minimum allowable catapulted take-off speed is calculated. This speed allows determining an allowable trajectory angle of the incline for the rectilinear altitude gain. The pressure distribution over the outer surface of the fuselage and the wing consoles of an unmanned aerial vehicle is obtained. The value of the acting axial force is calculated for take-off modes with different angles of inclination of the trajectory and for a horizontal straight-line flight at a constant speed.
PDF
