Light-induced alterations in cell shape and pigment displacement in chromatophores of the sea urchin Centrostephanus longispinus

Summary Alterations in cell shape of the light-sensitive chromatophores of Centrostephanus longispinus are described. Upon illumination a centrifugal pigment movement starts within extremely thin filopodia which radiate from the cell body. With continued pigment migration the cellular processes increase in length and diameter and give the cell an irregular stellate appearance. Pigment movement within the cellular processes is discontinuous in space and time and may occur independently in single filopodia. The motion of single granules shows characteristic features of a saltatory movement.     

Chemical genetics: tailoring tools for cell biology

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.     

Chemical cytometry

Chemical cytometry refers to the use of high-sensitivity analytical tools to characterize single cells. These tools include mass spectrometry, electrochemistry and capillary separation methods. This review focuses on the use of capillary electrophoresis coupled with high-sensitivity detection to characterize single cells. In survey experiments, biogenic amines and proteins have been characterized in single cells. In directed experiments, fluorescent substrates are used to monitor the activity of sets of enzymes, either within a family or along an enzymatic cascade. When combined with classical cytometry tools, it is now possible to monitor several cellular components in single cells as a function of cell cycle, which provides insight into the evolution of cellular composition as cells prepare for division.     

Molecular live cell bioimaging in stem cell research

Functional heterogeneity within stem and progenitor cells has been shown to influence cell fate decisions. Similarly, intracellular signaling activated by external stimuli is highly heterogeneous and its spatiotemporal activity is linked to future cell behavior. To quantify these heterogeneous states and link them to future cell fates, it is important to observe cell populations continuously with single cell resolution. Live cell imaging in combination with fluorescent biosensors for signaling activity serves as a powerful tool to study cellular and molecular heterogeneity and the long-term biological effects of signaling. Here, we describe these methodologies, their advantages over classical approaches, and we illustrate how they could be applied to improve our understanding of the importance of heterogeneous cellular and molecular responses to external signaling cues.     

Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well‐established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi‐omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two‐phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10–15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time‐resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in‐depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.     

Cellular models to investigate biochemical pathways in Parkinson's disease

Cellular models are instrumental in dissecting a complex pathological process into simpler molecular events. Parkinson's disease is multifactorial and clinically heterogeneous; the aetiology of the sporadic (and most common) form is still unclear and only a few molecular mechanisms have been clarified so far in the neurodegenerative cascade. In such a multifaceted picture, it is particularly important to identify experimental models that simplify the study of the different networks of proteins/genes involved. Cellular models that reproduce some of the features of the neurons that degenerate in Parkinson's disease have contributed to many advances in our comprehension of the pathogenic flow of the disease. In particular, the pivotal biochemical pathways (i.e. apoptosis and oxidative stress, mitochondrial impairment and dysfunctional mitophagy, unfolded protein stress and improper removal of misfolded proteins) have been widely explored in cell lines, challenged with toxic insults or genetically modified. The central role of α-synuclein has generated many models aiming to elucidate its contribution to the dysregulation of various cellular processes. In conclusion, classical cellular models appear to be the correct choice for preliminary studies on the molecular action of new drugs or potential toxins and for understanding the role of single genetic factors. Moreover, the availability of novel cellular systems, such as cybrids or induced pluripotent stem cells, offers the chance to exploit the advantages of an in vitro investigation, although mirroring more closely the cell population being affected.     

Regulation of bacterial physiological processes by three types of protein phosphorylating systems

A single type of protein-phosphorylating system, the ATP-dependent protein kinases, is employed in the regulation of a variety of cellular physiological processes in eukaryotes. By contrast, recent work with bacteria has revealed that three types of protein-phosphorylating systems are involved in regulation: (1) the classical protein kinases, (2) the newly discovered sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological and mechanistic aspects of these three evolutionarily distinct systems are discussed.     

Self-maintained escort cells form a germline stem cell differentiation niche

Stem cell self-renewal is controlled by concerted actions of niche signals and intrinsic factors in a variety of systems. In the Drosophila ovary, germline stem cells (GSCs) in the niche continuously self-renew and generate differentiated germ cells that interact physically with escort cells (ECs). It has been proposed that escort stem cells (ESCs), which directly contact GSCs, generate differentiated ECs to maintain the EC population. However, it remains unclear whether the differentiation status of germ cells affects EC behavior and how the interaction between ECs and germ cells is regulated. In this study, we have found that ECs can undergo slow cell turnover regardless of their positions, and the lost cells are replenished by their neighboring ECs via self-duplication rather than via stem cells. ECs extend elaborate cellular processes that exhibit extensive interactions with differentiated germ cells. Interestingly, long cellular processes of ECs are absent when GSC progeny fail to differentiate, suggesting that differentiated germ cells are required for the formation or maintenance of EC cellular processes. Disruption of Rho functions leads to the disruption of long EC cellular processes and the accumulation of ill-differentiated single germ cells by increasing BMP signaling activity outside the GSC niche, and also causes gradual EC loss. Therefore, our findings indicate that ECs interact extensively with differentiated germ cells through their elaborate cellular processes and control proper germ cell differentiation. Here, we propose that ECs form a niche that controls GSC lineage differentiation and is maintained by a non-stem cell mechanism.     

Fish bone‐derived cell lines: an alternative in vitro cell system to study bone biology

Human bone diseases represent a major health problem worldwide and effective therapies have still to be developed. Despite numerous studies using mammalian systems, cellular and molecular processes governing bone and cartilage homeostasis in vertebrates are still not fully understood. Recently, fish have emerged as a suitable model and a promising alternative to the classical mammalian systems to study vertebrate development, in particular skeletogenesis. To complement in vivo developmental studies and identify signalling pathways involved in development processes, fish cell lines have been developed, in particular bone‐derived cells. This work intends to review what is presently known about fish bone‐derived cell lines, focusing on their relevance for bone biology studies.     

Single molecule microscopy of biomembranes (review)

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.     

Single molecule microscopy of biomembranes (Review)

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

Novel Chimeric Peptide Inhibits Protein Kinase C and Induces Apoptosis in Human Immune Cells

Protein kinase C (PKC) participates in a myriad of cellular processes. Protein kinase C isoforms play different roles based on their cellular expression balance and activation. The activity of classical PKC isoforms has been shown to be crucial for immune cell population homeostasis, playing a positive role in survival and proliferation. Protein kinase C inhibitors have been used for conditions where up-regulated PKC results in a pathological state. The most commonly investigated PKC inhibitors are highly effective in inhibiting PKC function but they are relatively unspecific, some of them even inhibiting other kinase families. Protein kinase C pseudosubstrates are auto-inhibitory domains which have been used to inhibit more specifically PKC in vitro but they do not freely penetrate cells. This could be resolved by using cell-permeable PKC pseudosubstrates which would more accurately modulate cellular PKC activity and PKC-related functions in intact cells. Here we show the development of a chimeric peptide inhibitor of classical PKC isoforms, consisting of a cell permeable sequence and a pseudosubstrate sequence which was able to translocate into cells, inhibiting PKC kinase activity and PKC T-cell-specific substrate phosphorylation. We also demonstrate a dramatic reduction in T-cell proliferation at high chimeric peptide concentration; this was attributed to apoptosis induction, as demonstrated by cell shrinking, phosphatidylserine exposure and DNA fragmentation. As expected, the control peptide (pseudosubstrate) did not penetrate cells, affect cell proliferation or survival. We also show that a neoplastic T-cell line which expresses higher levels of PKC is more resistant to chimeric peptide-mediated cell death than normal cells, corroborating a PKC role in apoptosis resistance. This chimeric peptide could be useful for the specific modulation of the PKC signalling pathway in pathological conditions.     

A monomeric myosin VI with a large working stroke

Myosin VI is involved in a wide variety of intracellular processes such as endocytosis, secretion and cell migration. Unlike almost all other myosins so far studied, it moves towards the minus end of actin filaments and is therefore likely to have unique cellular properties. However, its mechanism of force production and movement is not understood. Under our experimental conditions, both expressed full-length and native myosin VI are monomeric. Electron microscopy using negative staining revealed that the addition of ATP induces a large conformational change in the neck/tail region of the expressed molecule. Using an optical tweezers-based force transducer we found that expressed myosin VI is nonprocessive and produces a large working stroke of 18 nm. Since the neck region of myosin VI is short (it contains only a single IQ motif), it is difficult to reconcile the 18 nm working stroke with the classical 'lever arm mechanism', unless other structures in the molecule contribute to the effective lever. A possible model to explain the large working stroke of myosin VI is presented.     

METHODS FOR GENTLE, DIFFERENTIAL HEATING OF PART OF A SINGLE LIVING CELL

The fabrication and use of resistance wire microheaters is described and the results obtainable with cells are illustrated. The microheaters permit localized warming of a small area, wherever desired, within a single living cell. This is directly shown by the production of localized structural changes in the mitotic spindle. Thus in this case, subcellular areas respond independently to the local temperature. The method is applicable to a variety of cellular processes, providing clues to cellular control mechanisms.     

Invasion and migration of a single chick pre-streak stage epiblast cell in vitro: Its implication to the primitive streak formation

To investigate the contribution of the epiblast cell behavior to the primitive streak formation, we examined the motility of a single epiblast cell from pre-streak stage embryo in vitro . On the substratum that was evenly coated with laminin gel, epiblast cells attached well to the gel and one or a few very long and broad cellular processes protruded from their spherical cell bodies; however, they hardly locomoted on it. Unexpectedly, after overnight culture, half of the single cells dissolved the laminin gel beneath them to make well-like holes, and invaded in the holes. On the substratum lined parallel with the fibrous laminin gels supplemented with fibronectin, they locomoted actively in accordance with the alignment. That is, they were subjected to contact guidance. In locomotion they looked like snails, extending one or a few long and broad processes in a forward direction from the spherical cell bodies. However, on the substratum lined with laminin or fibronectin only, they did not locomote actively. Individual chick pre-streak epiblast cells had already been committed to invade, and their migratory nature existed in each cell, even though they were isolated from the epithelial sheet. The implication of these findings on the cellular basis of primitive streak formation will be discussed.