Three-dimensional imaging of fast intracellular processes by fluorescence microscopy should provide decent spatial and high temporal resolution while minimizing fluorophore bleaching and ...cytotoxicity. We give a condensed introductory overview of three contemporary methods mostly used for imaging of living cells in 3D and compare their performance in terms of temporal and spatial resolution, imaging flexibility and specimen photodamage: point-scanning confocal microscopy, spinning-disc confocal microscopy, and lattice light-sheet microscopy. While point-scanning instruments are unsurpassed in terms of confocal performance, flexibility and configurability of their optical path, spinning-disc and lattice light-sheet optical designs excel in acquisition speed and low levels of light-inflicted specimen deterioration.
Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material ...by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.
While a small proportion of high‐risk (HR) alpha (α) human papillomaviruses (HPVs) is associated with numerous human malignancies, of which cervical cancer is the most prevalent, beta (β) HPVs ...predominantly act as co‐factors in skin carcinogenesis. A characteristic feature of both α‐ and β‐E6 oncoproteins is the presence of the LXXLL binding motif, which α‐E6s utilize to form a complex with E6AP and which enables β‐E6s to interact with MAML1. Here we show that multiple α‐E6 oncoproteins bind to MAML1 via the LXXLL binding motif and that this results in increased protein stability. Moreover, β‐E6 oncoprotein stability is also dependent on the interaction with MAML1. Additionally, in the absence of MAML1, endogenous HPV‐8 E6 and HPV‐18 E6 are rapidly degraded at the proteasome. Ablation of both E6AP and MAML1 leads to an even more profound downregulation of α‐E6 protein expression, whereas this is not observed with β‐E6. This highly suggests that there is one cellular pool for most of β‐E6 that interacts solely with MAML1, whereas there are two cellular pools of HR α‐E6, one forming a complex with MAML1 and the other interacting with E6AP. Furthermore, MAML1 induces HPV‐8 E6 shuttling from the nucleus to the cytosolic fraction, while MAML1 interaction with HR E6 induces a drastic nuclear and membrane upregulation of E6. Interestingly, the HR α‐E6/MAML1 complex does not affect targeting of some of the known HR E6 cellular substrates such as p53 and DLG1. However, MAML1 and E6AP joint co‐expression with HR α‐E6 leads to a significant increase in cellular proliferation, whereas silencing MAML1 decreases wound closure in HeLa cells. These results demonstrate that HR α‐E6 interaction with MAML1 results in a stable form of E6, which likely modulates MAML1's normal cellular activities, one consequence of which being an increased proliferative capacity of HPV‐transformed cancer cells. Thus, this study shows a novel function of the α‐E6 oncoprotein and how it's activity might affect HPV‐induced pathogenesis.
NME proteins are reported to influence signal transduction activity of small GTPases from the Ras superfamily by diverse mechanisms in addition to their generic NDP kinase activity, which replenishes ...the cytoplasmic pool of GTP. Comprehensive evidence shows that NME proteins modulate the activity of Ras GTPases, in particular members of the Rho family, via binding to their major activators GEFs. Direct interaction between several NMEs and Ras GTPases were also indicated in vitro and in vivo. These modes of regulation are mainly independent of the NME's kinase activity. NMEs also modulate the Ras-mediated signal transduction by interfering with the formation of a Ras signaling complex at the plasma membrane. In several examples, NMEs were proposed to perform the role of GAP proteins by promoting hydrolysis of the bound GTP, but this activity still requires additional verification. Early suggestions that NMEs can activate small GTPases by direct phosphorylation of the bound GDP, or by high-rate loading of GTP onto a closely apposed GTPase, were largely dismissed. In this review article, we survey and put into perspective published examples of identified and hypothetical mechanisms of Ras signaling modulation by NME proteins. We also point out involvement of NMEs in the transcriptional regulation of components of Ras GTPases-mediated signal transduction pathways, and reciprocal regulation of NME function by small GTPases, particularly related to NME's binding to membranes.
Both
amoebae and mammalian cells are endowed with an elaborate actin cytoskeleton that enables them to perform a multitude of tasks essential for survival. Although these organisms diverged more than ...a billion years ago, their cells share the capability of chemotactic migration, large-scale endocytosis, binary division effected by actomyosin contraction, and various types of adhesions to other cells and to the extracellular environment. The composition and dynamics of the transient actin-based structures that are engaged in these processes are also astonishingly similar in these evolutionary distant organisms. The question arises whether this remarkable resemblance in the cellular motility hardware is accompanied by a similar correspondence in matching software, the signalling networks that govern the assembly of the actin cytoskeleton. Small GTPases from the Rho family play pivotal roles in the control of the actin cytoskeleton dynamics. Indicatively,
matches mammals in the number of these proteins. We give an overview of the Rho signalling pathways that regulate the actin dynamics in
and compare them with similar signalling networks in mammals. We also provide a phylogeny of Rho GTPases in Amoebozoa, which shows a variability of the Rho inventories across different clades found also in Metazoa.
Since the role of the major mitochondrial NAD+-dependent deacetylase, sirtuin 3 (Sirt3), is differential in cancer, opposite to the well-known tumor-suppressing effect of hyperoxia, this study aimed ...to investigate the role of Sirt3 in triple-negative breast cancer (TNBC) cell line MDA-MB-231 upon hyperoxic (95% O2) conditions.
MDA-MB-231 cells were stably transfected with Flag-tagged Sirt-3 or empty plasmid. Western blot and real-time PCR were used to monitor the expression of proteins or genes involved in mitochondrial biogenesis, metabolic regulation and antioxidant defense. Immunocytochemistry and confocal microscopy were used to confirm the cellular localization and abundance of proteins. Flow cytometry was used to analyze mitochondrial mass, potential and ROS production, and MTT test as a measure of metabolic activity. Mitotic index analysis, colony-forming unit assay, DNA damage and Annexin V-FITC analyses were used to assess the differences in the growth and apoptosis rate.
Although Sirt3 seemed to improve mitochondrial properties by increasing mitochondrial mass and potential, metabolic activity (Warburg effect) and antioxidative defense (SOD2, Cat), it also increased mitochondrial ROS, induced DNA damage, timp-1 expression, formation of multinucleated cells and apoptosis, and finally markedly reduced the proliferation of MDA-MB-231 cells. All these effects were even more evident upon the hyperoxic treatment, thus pointing towards combined negative effect of Sirt3 and hyperoxia on MDA-MB-231 cells.
Both Sirt3 and hyperoxia, alone or in combination, have the potential to negatively affect the malignant properties of the MDA-MB-231 cells and should be further explored as a possible therapy for TNBC.
•A novel RecBCD- RecFOR-independent recombination pathway is identified in E. coli.•RecBCD- RecFOR-independent (RecBFI) pathway is active in sbcB15 sbcC(D) mutants.•The pathway is very efficient in ...transduction, and less efficient in DNA repair.•recA, recJ and recQ gene functions are essential for RecBFI pathway.•Mutant SbcB15 protein might be involved in RecA-loading during RecBFI recombination.
The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB+ sbcC+ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.
Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, ...a fluorescent probe was developed that specifically binds to the GTP-bound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.
Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on ...fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation, and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile
Dictyostelium
cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase and was selected on the basis of yeast two-hybrid screen, GST pull-down assay and FRET measurements by fluorescence lifetime imaging microscopy. DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison with similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis.
We describe a simple optical configuration for dark-field microscopy at low magnification, realized with the use of standard microscope components. An inherent high contrast makes this method ...attractive for computer-assisted tracking and counting of microorganisms. We applied this setup for dark-field microscopy to measure the speed of migrating Dictyostelium amoebae.
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•A standard microscope was adapted for low-magnification dark-field microscopy.•Optical resolution of the dark-field setup is 2.8μm.•We used this setup for computer-assisted tracking of Dictyostelium amoebae.•High-contrast dark-field images of bacteria and yeast cells are shown.
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