Dancing with the Stars: Using Image Analysis to Study the Choreography of the Endoplasmic Reticulum and Its Partners and of Movement Within Its Tubules.

In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or non-moving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topologies, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to non-moving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that move rapidly and stream through the cell, image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.

The Sterol Trafficking Pathway in Arabidopsis thaliana.

In plants, the trafficking mechanisms by which sterols move through the plant and into target cells are unknown. Earlier studies identified endosomes as primary candidates for internalization of sterols in plants, but these results have come into question. Here, we show that in elongating root cells, the internalization of sterol occurs primarily by a non-endocytic mechanism. Added fluorescent sterols [dehydroergosterol (DHE) and BODIPY-cholesterol (BCh)] do not initially label endosomes identified by fluorescent protein markers or by internalized FM4-64. Instead, the nuclear envelope, an organelle not associated with the endocytic pathway but part of the endoplasmic reticulum (ER), becomes labeled. This result is supported by experiments with the inducible overexpression of auxilin-2-like protein (AUX2 line), which blocks most endocytosis upon induction. Internalization and nuclear envelope labeling still occur in induced AUX2 cells. Longer-term incubation labels the oil body, a site involved in sterol storage. Although the first site of localization, the nuclear envelope, is part of the ER, other domains of the ER do not accumulate the label. The trafficking pathway differs from vesicular endocytosis and points toward a different pathway of sterol transport possibly involving other mechanisms, such as ER-plasma membrane contact sites and cytoplasmic transport.

The lost portrait of Robert Hooke?

This letter considers the 'Portrait of a Mathematician' attributed to Mary Beale in the 1680s as a likely candidate for a portrait of Robert Hooke made during his lifetime. It closely matches the physical descriptions of Hooke made by his biographers who knew him, Richard Waller (d. 1715) and John Aubrey (1626-1697). The portrait contains a remarkable diagram, as well as its mechanical analogue, demonstrating the elliptical orbit of a body under constant force similar to an unpublished, unfinished 1685 manuscript by Hooke. It also contains a landscape, which is likely to be Lowther Castle and its associated church. Hooke provided designs for the Lowther castle church renovations completed in 1686. The portraitist, Mary Beale, knew Hooke and was very familiar with the Lowther family, who commissioned 30 portraits from her. The diagram and armillary sphere in the portrait establish Hooke's contribution to the theory of gravity. Their presence may have been one of the reasons the portrait was not purchased and kept by the Royal Society. It might have diminished the legacy of Isaac Newton.

Dancing with the Stars: Using Image Analysis to Study the Choreography of the Endoplasmic Reticulum and Its Partners and of Movement Within Its Tubules.

In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or nonmoving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topology, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to nonmoving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that are moving rapidly and streaming through the cell, the image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.

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.

ER network dynamics are differentially controlled by myosins XI-K, XI-C, XI-E, XI-I, XI-1, and XI-2.

The endoplasmic reticulum (ER) of higher plants is a complex network of tubules and cisternae. Some of the tubules and cisternae are relatively persistent, while others are dynamically moving and remodeling through growth and shrinkage, cycles of tubule elongation and retraction, and cisternal expansion and diminution. Previous work showed that transient expression in tobacco leaves of the motor-less, truncated tail of myosin XI-K increases the relative area of both persistent cisternae and tubules in the ER. Likewise, transient expression of XI-K tail diminishes the movement of organelles such as Golgi and peroxisomes. To examine whether other class XI myosins are involved in the remodeling and movement of the ER, other myosin XIs implicated in organelle movement, XI-1 (MYA1),XI-2 (MYA2), XI-C, XI-E, XI-I, and one not, XI-A, were expressed as motor-less tail constructs and their effect on ER persistent structures determined. Here, we indicate a differential effect on ER dynamics whereby certain class XI myosins may have more influence over controlling cisternalization rather than tubulation.

Who invented the dichotomous key? Richard Waller's watercolors of the herbs of Britain.

On 27 March 1689, Richard Waller, Fellow and Secretary of the Royal Society presented his "Tables of the English Herbs reduced to such an order, as to find the name of them by their external figures and shapes" to his assembled colleagues at a meeting of the Royal Society. These tables were developed for the novice by being color images, composed in pencil and watercolor, of selected plants and their distinguishing characteristics. The botanical watercolors for the tables are now a Turning-the-Pages document online on the website of the Royal Society. However, for the past 320 years, the scientific context for the creation of these outstanding botanical watercolors has remained obscure. These tables were developed by Waller as an image-based dichotomous key, pre-dating by almost 100 years the text-based dichotomous keys in the first edition of Flora Française (1778) by Jean Baptiste Lamarck, who is generally given priority for the development of the dichotomous key. How these large folio images were arranged to illustrate a dichotomous key is unknown, but an arrangement based on Waller's description is illustrated here as leaf-ordering for the separate hierarchical clusters (tables). Although only 24 species of watercolored dicot herbs out of a total of 65 in the set of watercolors (the others being monocots) are used in these tables, they are a "proof of concept", serving as models upon which a method is based, that of using a key composed of dichotomous choices for aiding identification.

Laser stimulation of the chloroplast/endoplasmic reticulum nexus in tobacco transiently produces protein aggregates (boluses) within the endoplasmic reticulum and stimulates local ER remodeling.

Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER? In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm (3-6-mW) laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates (boluses) within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45°C, temperature (heat stress). Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.

Networking in the endoplasmic reticulum.

The network of the ER (endoplasmic reticulum) is set up by cytoskeletal control of the movement and remodelling of polygonal rings of tubules, bundles of tubules and cisternal regions. We have developed a new image analysis tool, persistency mapping, to understand the framework upon which the plant ER remodels. With this new tool, we have explored the network nodes, called anchor/growth sites, that may anchor the network by attachment to the plasma membrane. We have determined how the polygonal ring structure depends on myosin XI-K for 'opening' and 'closing'. With latrunculin B treatment, we have investigated the involvement of actin in the elongation and persistency of the tubules. We also show how the cytoskeleton is involved in directional diffusion within the membrane. This observation may lead to an answer to the question of what function this network structure serves in the cell. We propose that the ER acts as a trafficking network, delivering lipid, protein, calcium and signalling molecules to different regions of the cell. It does so by directional reduced dimensional diffusion. The ER network of tubules restricts the dimensionality of diffusion to near one-dimensional, whereas the cisternae reduce it to near two-dimensional. The cytoskeleton does not drive the diffusion, but participates by providing directionality to the diffusion.