Proteomics of regulated secretory organelles

Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles‐proteomic analysis is discussed as well. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:844–867, 2009     

Fluorescent proteins as markers in the plant secretory pathway

The use of fluorescent proteins and live cell imaging has greatly increased our knowledge of cell biology in recent years. Not only can these technologies be used to study protein trafficking under different conditions, but they have also been of use in elucidating the relationships between different organelles in a noninvasive manner. The use of multiple different fluorochromes allows the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields at this time. In this review, we discuss the multitude of fluorescent markers that have been generated to study the plant secretory pathway. Although these markers have been used to solve many mysteries in this field, some areas that require further discussion remain.     

Investigation of the morphology, viability and mechanical properties of yeast cells in environmental SEM

The mechanical properties of biological cells at nanoscale may be characterized using an environmental scanning electron microscopy (ESEM) combined with a force measurement device. However, the electron beam radiation in an ESEM may damage a specimen. So far, little is known about the radiation damage to biological cells. In this work, single yeast cells were imaged using an ESEM under both high and low vacuum modes. The changes in their morphology and viability were monitored as a function of radiation time for a given beam current of 538 pA corresponding to 10 kV accelerating voltage and spot size 4. Under the two modes, the radiation damage to the morphology of yeast cells became evident after an exposure time of 3 min, but under the low vacuum mode, the damage to their morphology was more severe. However, all cells lost their viability after 5 min under the high vacuum mode with the electron beam off from an initial viability of 95+/-1%. In contrast, the viability of cells under the low vacuum mode was found to be approximately 20% after 20 min. In addition, a newly developed ESEM-based nanomanipulation technique was applied to measure the force imposed on single yeast cells and their deformation, including contact diameter and central lateral diameter for the compression of single yeast cells to a given displacement within a time frame of 1 min, and the data obtained may be used to validate mathematical modeling of the stress-strain relationship for the compression of cells in order to determine their intrinsic mechanical property parameters.     

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

A combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell.     

RHO GTPases in the control of cell morphology, cell polarity, and actin localization in fission yeast.

The fission yeast Schizosaccharomyces pombe undergoes morphogenetic changes during both vegetative and sexual cell cycles that require asymmetric cell growth and actin cytoskeleton reorganisations. Different complex signal transduction pathways participate in S. pombe morphogenesis. The Rho family of GTPases are present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signalling pathways that control actin organisation and morphogenetic processes is well known. In this review, we will briefly summarize the role of the Rho GTPases in the establishment and maintenance of cell polarity and growth of S. pombe. As in other fungi, S. pombe morphogenesis is closely related to cell wall biosynthesis, and Rho GTPases are critical modulators of this process. They provide the coordinated regulation of cell wall biosynthetic enzymes and actin organisation required to maintain cell integrity and polarised growth.     

ER exit pathways and the control of proteostasis: Crucial role of the UPR,COPII, and ER-phagy in the secretory pathway

The endoplasmic reticulum (ER) is the site of entry of all proteins that function in the secretory pathway including the extracellular environment. Because it controls the folding of newly synthesized secretory proteins, the ER is indispensable for the maintenance of proteostasis in the secretory pathway. Within the ER and, in part, in post-ER compartments, the quality control of protein folding is under the regulation of the unfolded protein response (UPR) pathways. The UPR strategy is to enhance protein folding, increase the ER degradation pathway of misfolded proteins, and allow the exit from the ER of only correctly folded proteins. The latter is controlled by the multimeric complex COPII, which also provides some of the components for ER-phagy the only route for the disposal of protein aggregates. In this overview, we wish to contribute to the introduction of new perspectives in the study of the mechanisms underlying the control of proteostasis within the secretory pathway.     

Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.     

Functional morphology and structural characteristics of wings of the ladybird beetle,Coccinella septempunctata (L.)

In recent years, the surface morphology and microstructure of ladybird (Coccinella septempunctata) wings have been used to help design the flapping‐wing micro air vehicle (FWMAV). In this study, scanning electron microscopy (SEM) was used to verify the functional roles of the ladybird forewing and hindwing. Surface morphology and the cross‐sectional microstructure of the wings are presented. Detailed morphology of ladybird forewings was observed using atomic force microscopy (AFM) and the composition of the wings was characterized using Fourier transformed infrared spectroscopy (FTIR). The ladybird forewing may possess different performance characteristics than the beetle, Allomyrina dichotoma. Additionally, the circular holes in the forewing might be important for decreasing the weight of the forewing and to satisfy requirements of mechanical behavior. Microsc. Res. Tech. 79:550–556, 2016. © 2016 Wiley Periodicals, Inc.     

Identification of protein associations in organelles, using mass spectrometry-based proteomics

Recent literature that highlights the power of using mass spectrometry (MS) for protein identification from preparations of highly purified organelles and other large subcellular structures is covered in this review with an emphasis on techniques that preserve the integrity of the functional protein complexes. Recent advances in distinguishing contaminant proteins from "bonafide" organelle-localized proteins and the affinity capture of protein complexes are reviewed, as well as bioinformatic strategies to predict protein organellar localization and to integrate protein-protein interaction maps obtained from MS-affinity capture methods with data obtained from other techniques. Those developments demonstrate that a revolution in cellular biology, fueled by technical advances in MS-based proteomic techniques, is well underway.     

Fixation induces differential polarized translocations of organelles in hyphae of Saprolegnia ferax

Saprolegnia hyphal tips were examined during fixation, and living or freeze-substituted tips were quantitatively compared with those fixed in commonly employed formulations of paraformaldehyde and glutaraldehyde. Treating hyphae with fixatives induced extensive longitudinal translocations of the cytoplasm and organelles, usually beginning with contractions toward the tip. These translocations were minimal in the extreme apex (~10 μm) and more extensive subapically. Hypertonic media or hypotonic buffers seldom or never induced translocations, respectively; in contrast, hypotonic buffers containing detergents or the Ca²⁺ -ionophore, A23187, frequently induced contractions. All fixations caused net nucleus movement away from the tip, with the amount of displacement depending on the pre-fixation distance from the tip. Similarly, all fixations moved the most-apical of saltatory vesicles away from the tip, but the total number in the apex increased or decreased depending on the fixative used. The patterns of these results suggest that nucleus and vesicle distribution controls may be related (with respect to most-apical organelles) but also at least partially independent (with respect to organelle populations in hyphal tips). Hyphal diameter was reduced by some, but not all fixations; this variability did not correlate with displacements of either organelle, nor with fixative osmotic pressure. Evidently fixative-induced changes are more complex and systematic in highly polarized tip-growing cells than previously reported in other, less polarized, cell types. These results also suggest that hyphae contain multiple and complex organelle distribution and hyphal diameter control systems which can be readily altered, often subtly, by fixation protocols commonly and uncritically employed in immunocytochemical and ultrastructural analyses, and that fixation can cause serious cellular reorganization.     

Use of AFM for imaging and measurement of the mechanical properties of light-convertible organelles in plants

We obtained topographic images of etioplasts and chloroplasts and measured their elasticity in a physiological buffer using an atomic force microscope (AFM) and found a possible correlation between the morphological and mechanical properties during the light conversion of etioplasts to chloroplasts. Alcian blue 8GX dye was found to be effective for immobilizing the plant organelles stably on a glass surface for AFM experiments. We employed the tapping-mode AFM with a cantilever soft enough to measure the elasticity of the organelles in a liquid solution. The best images of soft, spherical organelles were obtained using the tapping-mode AFM with oscillation at the thermal vibration frequency of the cantilever of around 3 kHz. Whereas etioplasts were found to be smooth-surfaced and stiff against compression by the AFM tip, before light conversion to chloroplasts, they became rough-surfaced and mechanically soft after exposure to light. The elasticity of etioplasts was 20 times higher than that of chloroplasts, probably reflecting changes in their inner structures.     

Video microscopy of dynamic plant cell organelles: principles of the technique and practical application

Video microscopy, including video-enhanced contrast, ultraviolet and video-intensified fluorescence microscopy, was applied to the visualization and analysis of organelles and cytoskeletal elements at the border of resolution of the light microscope. We describe the principles of video microscopy and the necessary technical equipment, and discuss the advantages and limitations with the example of three selected plant cells. In characean internodal cells we observed the motility and disappearance of Golgi secretory vesicles during wound wall formation by video-enhanced contrast microscopy. In Byblis gland hairs we investigated the movement of different organelles along bundles of actin microfilaments by ultraviolet microscopy, and in onion inner epidermal cells we visualized the arrangement of actin microfilaments during different stages of plasmolysis with video-intensified fluorescence microscopy.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Statistical analysis of the quantal basis of secretory granule formation

ABSTRACT The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi‐modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping‐together artifact of the measurement or an inherent intra‐cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra‐cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM‐algorithm‐based method is presented for the conversion of multi‐modal to quantal data that provides as by‐product estimates of means and variances of basic granule packaging. Microsc. Res. Tech. 77:1–10, 2014. © 2013 Wiley Periodicals, Inc.