scholarly journals Overcoming efflux of fluorescent probes for actin imaging in living cells

2020 ◽  
Author(s):  
Rūta Gerasimaitė ◽  
Jan Seikowski ◽  
Jens Schimpfhauser ◽  
Georgij Kostiuk ◽  
Tanja Gilat ◽  
...  
Keyword(s):  
Cell Shape ◽  
Fruit Fly ◽  
Cell Types ◽  
Primary Neurons ◽  
Cellular Functions ◽  
Active Efflux ◽  
Wide Range ◽  
Preferential Binding ◽  
Membrane Pumps ◽  
2D And 3D

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

scholarly journals Signaling roles of phosphoinositides in the retina

2020 ◽  
pp. jlr.TR120000806 ◽  
Author(s):  
Raju V. S. Rajala
Keyword(s):  
Cell Types ◽  
Cellular Functions ◽  
Parent Molecule ◽  
Phosphoinositide Signaling ◽  
Wide Range ◽  
The Many ◽  
And Function ◽  
Different Cell Types

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

scholarly journals The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

2020 ◽  
Author(s):  
Paymaan Jafar-nejad ◽  
Berit Powers ◽  
Armand Soriano ◽  
Hien Zhao ◽  
Daniel A Norris ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Muscular Atrophy ◽  
Neurological Diseases ◽  
Cell Types ◽  
Rnase H ◽  
Central Administration ◽  
Spinal Motor Neurons ◽  
New Class ◽  
Wide Range ◽  
Therapeutic Development

Abstract Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

scholarly journals Energetics of mesoscale cell turbulence in two-dimensional monolayers

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shao-Zhen Lin ◽  
Wu-Yang Zhang ◽  
Dapeng Bi ◽  
Bo Li ◽  
Xi-Qiao Feng
Keyword(s):  
Kinetic Energy ◽  
Tumor Metastasis ◽  
Cell Types ◽  
Boltzmann Distribution ◽  
Scaling Exponent ◽  
Biological Processes ◽  
Two Dimensional ◽  
Cell Monolayers ◽  
Wide Range ◽  
Epithelial Cell Monolayers

AbstractInvestigation of energy mechanisms at the collective cell scale is a challenge for understanding various biological processes, such as embryonic development and tumor metastasis. Here we investigate the energetics of self-sustained mesoscale turbulence in confluent two-dimensional (2D) cell monolayers. We find that the kinetic energy and enstrophy of collective cell flows in both epithelial and non-epithelial cell monolayers collapse to a family of probability density functions, which follow the q-Gaussian distribution rather than the Maxwell–Boltzmann distribution. The enstrophy scales linearly with the kinetic energy as the monolayer matures. The energy spectra exhibit a power-decaying law at large wavenumbers, with a scaling exponent markedly different from that in the classical 2D Kolmogorov–Kraichnan turbulence. These energetic features are demonstrated to be common for all cell types on various substrates with a wide range of stiffness. This study provides unique clues to understand active natures of cell population and tissues.

scholarly journals Antimicrobial Nodule-Specific Cysteine-Rich Peptides Induce Membrane Depolarization-Associated Changes in the Transcriptome of Sinorhizobium meliloti

2013 ◽  
Vol 79 (21) ◽  
pp. 6737-6746 ◽  
Author(s):  
Hilda Tiricz ◽  
Attila Szűcs ◽  
Attila Farkas ◽  
Bernadett Pap ◽  
Rui M. Lima ◽  
...  
Keyword(s):  
Stress Responses ◽  
Sinorhizobium Meliloti ◽  
Antimicrobial Agents ◽  
Cellular Functions ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Peptide Family ◽  
Wide Range ◽  
Natural Target

ABSTRACTLeguminous plants establish symbiosis with nitrogen-fixing alpha- and betaproteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the inverted repeat-lacking clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had antirhizobial activity measured by reduction or elimination of the CFU and uptake of the membrane-impermeable dye propidium iodide. Here, the antimicrobial spectrum of two of these peptides, NCR247 and NCR335, was investigated, and their effect on the transcriptome of the natural targetSinorhizobium melilotiwas characterized. Both peptides were able to kill quickly a wide range of Gram-negative and Gram-positive bacteria; however, their spectra were only partially overlapping, and differences were found also in their efficacy on given strains, indicating that the actions of NCR247 and NCR335 might be similar though not identical. Treatment ofS. meliloticultures with either peptide resulted in a quick downregulation of genes involved in basic cellular functions, such as transcription-translation and energy production, as well as upregulation of genes involved in stress and oxidative stress responses and membrane transport. Similar changes provoked mainly in Gram-positive bacteria by antimicrobial agents were coupled with the destruction of membrane potential, indicating that it might also be a common step in the bactericidal actions of NCR247 and NCR335.

scholarly journals A54 THE CIRCADIAN CLOCK INFLUENCES JAK/STAT SIGNALING AND GUT PERMEABILITY

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 11-12
Author(s):  
K Parasram ◽  
D Bachetti ◽  
P Karpowicz
Keyword(s):  
Circadian Clock ◽  
Clock Cycle ◽  
Intestinal Barrier ◽  
Fruit Fly ◽  
Cell Types ◽  
Time Of Day ◽  
Acute Injury ◽  
Intestinal Barrier Function ◽  
Intestinal Immunity ◽  
Stat Signaling

Abstract Background The circadian clock is a 24-hour feedback loop that drives rhythms in behaviours and physiological processes. This molecular timekeeper consists of the transcription factors, Clock-Cycle, that drive expression of thousands of clock-controlled genes, with two of these, Period and Timeless, acting as negative regulators of Clock-Cycle. This fundamental mechanism was initially characterized in the fruit fly, Drosophila melanogaster (Nobel Prize in Physiology & Medicine, 2017), and is highly conserved in humans. The intestine, or midgut, of Drosophila, is also similar to the human small intestine consisting of similar cellular lineage, signaling pathways, and physiological functions. The lineage of the Drosophila intestine contains the same four cell types as humans: intestinal stem cells (ISCs), progenitors called enteroblasts, enterocytes and enteroendocrine cells. This simplified lineage as well as the genetic tools available, make Drosophila an ideal model for intestinal regeneration in health and disease. We have previously shown that the circadian clock is active in ISCs, EBs and ECs during both homeostatic and regenerating conditions. Furthermore, the circadian clock regulates the mitosis of ISCs under regenerating conditions. Aims We sought to uncover if Jak/STAT signaling, one of the key pathways involved in ISC proliferation in the Drosophila intestine, shows a circadian rhythm and if there is a time-of-day difference in the regenerative response. Methods To test whether the clock regulates Jak/STAT during acute injury, we developed an irradiation assay that does not affect survival but acutely disrupts intestinal barrier function. Results Using a dynamic reporter of Jak/STAT activity we show that Period circadian clock mutants have low Jak/STAT signaling and a leaky gut phenotype. Wildtype controls show time-dependent gut leakiness upon irradiation, which is higher and time-independent in Period mutants. The level of Jak/STAT response differs depending on the time of irradiation in the controls, but is higher at all times in the mutants. Conclusions The Jak/Stat pathway regulates intestinal immunity and epithelial cell proliferation in humans, thus playing a role in colorectal cancer and inflammatory bowel disease. Our results suggest Jak/Stat is controlled by the circadian clock, which has implications for intestinal recovery following medical treatments, including radiation therapy. Funding Agencies NRC

Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster

2001 ◽  
Vol 114 (1) ◽  
pp. 37-47 ◽  
Author(s):  
G. Crevel ◽  
H. Huikeshoven ◽  
S. Cotterill
Keyword(s):  
Cell Types ◽  
Drosophila Embryo ◽  
Cell Fractionation ◽  
General Function ◽  
Structural Protein ◽  
Chromosomal Structure ◽  
Binding Characteristics ◽  
Wide Range ◽  
Cellular Dna

We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism. The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non-dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31. These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.

Protein kinase C-α and the regulation of diverse cell responses

2017 ◽  
Vol 8 (3-4) ◽  
pp. 143-153 ◽  
Author(s):  
Rishi Kant Singh ◽  
Sanjay Kumar ◽  
Pramod Kumar Gautam ◽  
Munendra Singh Tomar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cellular Localization ◽  
Cellular Transformation ◽  
Cell Types ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Cell Responses ◽  
Kinase C ◽  
Cellular Compartments

AbstractProtein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

scholarly journals The AAA+ ATPase p97, a cellular multitool

2017 ◽  
Vol 474 (17) ◽  
pp. 2953-2976 ◽  
Author(s):  
Lasse Stach ◽  
Paul S. Freemont
Keyword(s):  
Atp Hydrolysis ◽  
Current Status ◽  
Cellular Functions ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Proteotoxic Stress ◽  
Binding Domains ◽  
Wide Range ◽  
Aaa Atpases ◽  
Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

scholarly journals A Dynamic Stochastic Model of Frequency-Dependent Stress Fiber Alignment Induced by Cyclic Stretch

2009 ◽  
Author(s):  
Hui-Ju Hsu ◽  
Chin-Fu Lee ◽  
Roland Kaunas
Keyword(s):  
Cell Shape ◽  
Principal Direction ◽  
Focal Adhesions ◽  
Cyclic Stretch ◽  
Cell Alignment ◽  
Cellular Functions ◽  
Fiber Alignment ◽  
Dynamic Stochastic ◽  
Dynamic Structures ◽  
Point Level

Actin stress fibers (SFs) are bundles of actin filaments anchored at each end via focal adhesions. Myosin-generated contraction leads to the development of tension, which extends SFs beyond their unloaded lengths. In human aortic ECs, the level of SF extension is maintained at a set-point level of ∼1.10 (1). SFs are also dynamic structures and their continuous assembly and disassembly is critical to cellular functions involving changes in cell shape. Further, deformation of the extracellular matrix perturbs SF extension, leading to compensatory responses such as the gradual alignment of SFs perpendicular to the principal direction of cyclic stretch. The extent of cell alignment has been shown to depend on the pattern of matrix stretch; however, it is unclear how cells distinguish between different patterns of stretch to determine their unique responses.

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