Organelle extensions in plant cells

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

Dynamic tubules extended from different organelles are integral components of the rapid subcellular response machinery involved in maintaining optimal working conditions within plant cells.     

Dynamic niches in the origination and differentiation of haematopoietic stem cells

Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic 'niche', or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche.     

The ins and outs of yeast vacuole trafficking

Summary Vacuoles are ubiquitous organelles in the fungal and plant kingdoms. They serve a variety of functions and are important for cell homeostasis. A constant turnover of proteins and membranes makes vacuoles dynamic organelles. Various transport pathways share the vacuole as their joint destination. The trafficking pathways are regulated independently. In yeast cells many components of the protein and membrane transport machinery are known. Recent years have seen much progress in our understanding of the protein-sorting pathways and the biogenesis of this organelle. Improvements of our understanding of the vesicular transport pathways and vacuolar membrane fusion are reviewed.     

The ins and outs of yeast vacuole trafficking

Vacuoles are ubiquitous organelles in the fungal and plant kingdoms. They serve a variety of functions and are important for cell homeostasis. A constant turnover of proteins and membranes makes vacuoles dynamic organelles. Various transport pathways share the vacuole as their joint destination. The trafficking pathways are regulated independently. In yeast cells many components of the protein and membrane transport machinery are known. Recent years have seen much progress in our understanding of the protein-sorting pathways and the biogenesis of this organelle. Improvements of our understanding of the vesicular transport pathways and vacuolar membrane fusion are reviewed.     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

Mobile factories: Golgi dynamics in plant cells

The plant Golgi apparatus plays a central role in the synthesis of cell wall material and the modification and sorting of proteins destined for the cell surface and vacuoles. Earlier perceptions of this organelle were shaped by static transmission electron micrographs and by its biosynthetic functions. However, it has become increasingly clear that many Golgi activities can only be understood in the context of its dynamic organization. Significant new insights have been gained recently into the molecules that mediate this dynamic behavior, and how this machinery differs between plants and animals or yeast. Most notable is the discovery that plant Golgi stacks can actively move through the cytoplasm along actin filaments, an observation that has major implications for trafficking to, through and from this organelle.     

Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells.Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.     

Gutsy moves in mice: cellular and molecular dynamics of endoderm morphogenesis

Despite the importance of the gut and its accessory organs, our understanding of early endoderm development is still incomplete. Traditionally, endoderm has been difficult to study because of its small size and relative fragility. However, recent advances in live cell imaging technologies have dramatically expanded our understanding of this tissue, adding a new appreciation for the complex molecular and morphogenetic processes that mediate gut formation. Several spatially and molecularly distinct subpopulations have been shown to exist within the endoderm before the onset of gastrulation. Here, we review findings that have uncovered complex cell movements within the endodermal layer, before and during gastrulation, leading to the conclusion that cells from primitive endoderm contribute descendants directly to gut.     

Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus

Rice blast disease is caused by the hemibiotrophic fungus Magnaporthe oryzae, which invades living plant cells using intracellular invasive hyphae (IH) that grow from one cell to the next. The cellular and molecular processes by which this occurs are not understood. We applied live-cell imaging to characterize the spatial and temporal development of IH and plant responses inside successively invaded rice (Oryza sativa) cells. Loading experiments with the endocytotic tracker FM4-64 showed dynamic plant membranes around IH. IH were sealed in a plant membrane, termed the extra-invasive hyphal membrane (EIHM), which showed multiple connections to peripheral rice cell membranes. The IH switched between pseudohyphal and filamentous growth. Successive cell invasions were biotrophic, although each invaded cell appeared to have lost viability when the fungus moved into adjacent cells. EIHM formed distinct membrane caps at the tips of IH that initially grew in neighboring cells. Time-lapse imaging showed IH scanning plant cell walls before crossing, and transmission electron microscopy showed IH preferentially contacting or crossing cell walls at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Analysis of biotrophic blast invasion will significantly contribute to our understanding of normal plant processes and allow the characterization of secreted fungal effectors that affect these processes.     

Dynamic imaging of mammalian neural tube closure

Neurulation, the process of neural tube formation, is a complex morphogenetic event. In the mammalian embryo, an understanding of the dynamic nature of neurulation has been hampered due to its in utero development. Here we use laser point scanning confocal microscopy of a membrane expressed fluorescent protein to visualize the dynamic cell behaviors comprising neural tube closure in the cultured mouse embryo. In particular, we have focused on the final step wherein the neural folds approach one another and seal to form the closed neural tube. Our unexpected findings reveal a mechanism of closure in the midbrain different from the zipper-like process thought to occur more generally. Individual non-neural ectoderm cells on opposing sides of the neural folds undergo a dramatic change in shape to protrude from the epithelial layer and then form intermediate closure points to “button-up” the folds. Cells from the juxtaposed neural folds extend long and short flexible extensions and form bridges across the physical gap of the closing folds. Thus, the combination of live embryo culture with dynamic imaging provides intriguing insight into the cell biological processes that mold embryonic tissues in mammals.     

Advances in qualitative and quantitative plant membrane proteomics

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.     

Deciphering the Golgi apparatus: from imaging to genes

The Golgi apparatus is a vital organelle in eukaryotic cells. It grabs and processes secretory materials synthesized by the endoplasmic reticulum (ER) before sorting them to their destination. The Golgi also receives materials from vacuoles/lysosomes and the plasma membrane for further recycling to other compartments within the cell (1) (Figure 1). Given the vital role of the Golgi in a cell, it is important to understand how this organelle attains and maintains its structural and functional integrity during the intense processes of membrane traffic. Despite an equally central role of the Golgi in membrane traffic in eukaryotes, the organization of this organelle has some unique features in each cell system. Therefore, the wealth of information available on the structure and activity of the Golgi in one system is not always directly transferable to others. However, certain morphological and functional aspects are common among cell systems. Therefore, studying the factors that regulate organelle biogenesis and organization of the Golgi apparatus is important in basic cell biology of eukaryotes and may also contribute to a better understanding of how different cell systems have evolved. In this study, we report on the identification of Golgi mutants in plant cells. We have developed a screen that is a promising strategy not only for the identification of genes responsible for the morphological and functional integrity of the plant Golgi but could also provide fundamental information on other multicellular systems for which the power of forward genetics cannot be exploited as easily as in Arabidopsis.     

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.     

Intercellular adhesion can be visualized using fluorescently labeled fibrosarcoma HT1080 cells cocultured with hematopoietic cell lines or CD34(+) enriched human mobilized peripheral blood cells

BACKGROUND: Intercellular contacts between adjacent cells migrating over each other are important in many cellular processes. However, it has been difficult to visualize and identify dynamic intercellular adhesions between migrating cells in situ. METHODS: Two fluorescent membrane dyes, PKH2 and PKH26 for staining HT1080 and hematopoietic cells and cell lines, and an automated fluorescence microscopy system were used to monitor intercellular adhesion. RESULTS: Cellular extensions connecting two or more adjacent cells were visualized, showing the intercellular adhesion between migrating cells for minutes and up to hours. After cells adhered to each other, followed by cell migration in different directions, cellular extensions were dragged from the pivotal contact points in different focal planes. CD34(+)-enriched mobilized peripheral blood cells and six hematopoietic cell lines showed intercellular connections in cocultures with HT1080. However, the frequency of intercellular connections was variable in different cocultures. A cell density of about 3.1 x 10(4) cells/cm(2) for both cell lines in cocultures provided an adequate number of cells in each field of view, showing up to four intercellular connections per 100 total cells plated. DISCUSSION: The tools derived from this study will open new areas of investigation for understanding the mechanism of the intercellular adhesion process.     

Actin organization and dynamics in filamentous fungi

Growth and morphogenesis of filamentous fungi is underpinned by dynamic reorganization and polarization of the actin cytoskeleton. Actin has crucial roles in exocytosis, endocytosis, organelle movement and cytokinesis in fungi, and these processes are coupled to the production of distinct higher-order structures (actin patches, cables and rings) that generate forces or serve as tracks for intracellular transport. New approaches for imaging actin in living cells are revealing important similarities and differences in actin architecture and organization within the fungal kingdom, and have yielded key insights into cell polarity, tip growth and long-distance intracellular transport. In this Review, we discuss the contribution that recent live-cell imaging and mutational studies have made to our understanding of the dynamics and regulation of actin in filamentous fungi.     

Plant ER geometry and dynamics: biophysical and cytoskeletal control during growth and biotic response

The endoplasmic reticulum (ER) is an intricate and dynamic network of membrane tubules and cisternae. In plant cells, the ER ‘web’ pervades the cortex and endoplasm and is continuous with adjacent cells as it passes through plasmodesmata. It is therefore the largest membranous organelle in plant cells. It performs essential functions including protein and lipid synthesis, and its morphology and movement are linked to cellular function. An emerging trend is that organelles can no longer be seen as discrete membrane-bound compartments, since they can physically interact and ‘communicate’ with one another. The ER may form a connecting central role in this process. This review tackles our current understanding and quantification of ER dynamics and how these change under a variety of biotic and developmental cues.     

Why should cell biologists study microbial pathogens?

One quarter of all deaths worldwide each year result from infectious diseases caused by microbial pathogens. Pathogens infect and cause disease by producing virulence factors that target host cell molecules. Studying how virulence factors target host cells has revealed fundamental principles of cell biology. These include important advances in our understanding of the cytoskeleton, organelles and membrane-trafficking intermediates, signal transduction pathways, cell cycle regulators, the organelle/protein recycling machinery, and cell-death pathways. Such studies have also revealed cellular pathways crucial for the immune response. Discoveries from basic research on the cell biology of pathogenesis are actively being translated into the development of host-targeted therapies to treat infectious diseases. Thus there are many reasons for cell biologists to incorporate the study of microbial pathogens into their research programs.