The molecular mechanisms underlying acrosome biogenesis elucidated by gene-manipulated mice

Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-golgi network and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasingly genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This Review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation.

STUDY OF THE COMPOSITION OF ESSENTIAL OIL ACHILLEA NOBILIS L. AND ITS ANTIBACTERIAL INFLU-ENCE ON CHANGE OF STAPHYLOCOCCUS AUREUS CELL ULTRASTRUCTURE

The component composition of the Achillea. nobilis essential oil, which grows in Azerbaijan, was studied for the first time by the gas-chromato-mass spectrometry method. In the essential oil, 35 components are identified, of which the predominant are artemisia ketone (23.706%), thujone 22.400%), 2-bornanone (6,367%), eudesm-7(11)-en-4-ol (6.321%), eucalyptol (4.507%), cubenol (3.317%), lavandulol (2.975%), β-thujone (2.933%), β-eudesmol (2.702%), methyl hinokiate (2.108%), terpinen-4-ol (1.715%), 1,2-longidione (1.313%), limonene-6-ol, pivalate (1.188%), neryl (S)-2-methylbutanoate (1.120%), caryophyllene oxide (1.014%). The effect of A. nobilis essential oil on the ultrastructural organization of bacteria (S. aureus) was studied using transmission electron microscopy. It was revealed that under the influence of essential oil, a violation of the plasma permeability of the plasma membrane is observed, accompanied by diffusion of the fine-grained osmiophilic precipitate of the destroyed cytoplasmic structures, which leads to the loss of the layered structure of the wall elements and to a significant compaction of the matrix in comparison with the control preparations. The death of bacterial cells was observed with complete destruction of the integrity of all structures that make up their wall. Along with this, on ultrathin sections, focal accumulations of dead bacterial cells are found, surrounded by structurally changed elements of their walls. The remains of destructively altered fragments of CP are found on various parts of unseparated bacterial cells. All of the above shows that the essential oil of A. nobilis has a pronounced antibacterial activity.

Molecular structure of stress granules and their role in the eukaryotic cell

Stress granules (SGs) are cytoplasmic structures found in eukaryotic cells, from yeast to human cells. They are made up of proteins, RNA and small ribosome subunits (40S). They arise as a result of the rapid shutdown of active protein biosynthesis in the cell, which is the result of the appearance of a stress factor. The mechanism of regulation of protein biosynthesis in response to stress takes place at two control nodes: (1) phosphorylation of the α subunit of the eIF2 factor as a result of the action of stress-recognizing kinases or by modulation of the mTOR pathway activity, which regulates the initiation of protein biosynthesis by the formation of a complex within the so-called cap structure. The protein arrest causes aggregation of the translation process components and other cell components (other proteins or mRNA molecules) into SGs. A lot of data indicates the active participation of SGs in metabolic processes, their control role over pro- and anti-apoptotic processes as well as in the development of cancer, neurodegenerative diseases and their defensive role in viral infections. Running title: Stress granules in the cell

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

A Simplified Method for the Histochemical Detection of Iron in Paraffin Sections: Intracellular Iron Deposits in Central Nervous System Tissue

Although all cells contain iron, most histochemical methods fail to reveal the presence of iron within many cells of the central nervous system (CNS), particularly neurons. Previously, a sensitive method was developed that limited the extraction of iron in paraffin sections, and this method revealed staining within neurons. However, the staining was often too robust making it difficult to discern discrete intracellular structures. In 1970, a study incorporated acetone in an iron histochemical procedure to facilitate the demarcation of staining features. In the present study, both acetone and limits to iron extraction were included in a simplified staining procedure. This procedure was applied to paraffin sections of CNS tissue from CISD2 deficient and littermate control mice. Discrete nuclear and cytoplasmic staining features were detected in all mice. Although widely present in neurons, punctate cytoplasmic staining was particularly prominent in large neurons within the hindbrain. Evaluation of extended depth of focus images, from serial focal planes, revealed numerous stained cytoplasmic structures. Additionally, the simplified staining procedure was applied to paraffin sections from Alzheimer’s disease and control cases. Despite suboptimal processing conditions compared to mouse tissue, discrete staining of cytoplasmic structures was revealed in some neurons, although many other neurons had nondescript staining features. In addition, initial findings revealed iron deposited within some vessels from patients with Alzheimer’s disease. In summary, since paraffin sections are commonly used for histological preparations, this simplified histochemical procedure could facilitate the study of iron in various CNS conditions by revealing staining details often missed by other procedures.

Emerging Roles of Long Noncoding RNAs in the Cytoplasmic Milieu

While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients

Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

mRNA with Mammalian Codon Bias Accumulates in Yeast Mutants with Constitutive Stress Granules

Stress granules and P bodies are cytoplasmic structures assembled in response to various stress factors and represent sites of temporary storage or decay of mRNAs. Depending on the source of stress, the formation of these structures may be driven by distinct mechanisms, but several stresses have been shown to stabilize mRNAs via inhibition of deadenylation. A recent study identified yeast gene deletion mutants with constitutive stress granules and elevated P bodies; however, the mechanisms which trigger its formation remain poorly understood. Here, we investigate the possibility of accumulating mRNA with mammalian codon bias, which we termed the model RNA, in these mutants. We found that the model RNA accumulates in dcp2 and xrn1 mutants and in four mutants with constitutive stress granules overlapping with P bodies. However, in eight other mutants with constitutive stress granules, the model RNA is downregulated, or its steady state levels vary. We further suggest that the accumulation of the model RNA is linked to its protection from the main mRNA surveillance path. However, there is no obvious targeting of the model RNA to stress granules or P bodies. Thus, accumulation of the model RNA and formation of constitutive stress granules occur independently and only some paths inducing formation of constitutive stress granules will stabilize mRNA as well.

mRNA with mammalian codon bias accumulates in yeast mutants with constitutive stress granules

Stress granules and P bodies are cytoplasmic structures assembled in response to various stress factors and represent sites of temporary storage or decay of mRNAs. Depending on the source of stress, the formation of these structures may be driven by distinct mechanisms, but several stresses were shown to stabilize mRNAs via inhibition of deadenylation. A recent study identified yeast gene deletion mutants with constitutive stress granules and elevated P bodies; however, the mechanisms which trigger its formation remain poorly understood. Here, we investigate the possibility of accumulating mRNA with mammalian codon bias, which we termed the model RNA, in these mutants. We found that the model RNA accumulates in dcp2 and xrn1 mutants and in four mutants with constitutive stress granules overlapping with P bodies. However, in eight other mutants with constitutive stress granules the model RNA is downregulated, or its steady state levels vary. We further suggest that the accumulation of the model RNA is linked to its protection from the main mRNA surveillance path. However, there is no obvious targeting of the model RNA to stress granules or P bodies. Thus, accumulation of the model RNA and formation of constitutive stress granules occur independently and only some paths inducing formation of constitutive stress granules will stabilize mRNA as well.