Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data.

Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components.

Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.

Signalling complexes and clusters: functional advantages and methodological hurdles.

Signalling molecules integrate, codify and transport information in cells. Organisation of these molecules in complexes and clusters improves the efficiency, fidelity and robustness of cellular signalling. Here, we summarise current views on how signalling molecules assemble into macromolecular complexes and clusters and how they use their physical properties to transduce environmental information into a variety of cellular processes. In addition, we discuss recent innovations in live-cell imaging at the sub-micrometer scale and the challenges of object (particle) tracking, both of which help us to observe signalling complexes and clusters and to examine their dynamic character.

In vivo localizations of membrane stress controllers PspA and PspG in Escherichia coli.

The phage shock protein (Psp) response in Gram-negative bacteria counteracts membrane stress. Transcription of the PspF regulon (pspABCDE and pspG) in Escherichia coli is induced upon stresses that dissipate the proton motive force (pmf). Using GFP fusions we have visualized the subcellular localizations of PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp). It has previously been proposed that PspA evenly coates the inner membrane of the cell. We now demonstrate that instead of uniformly covering the entire cell, PspA (and PspG) is highly organized into what appear to be distinct functional classes (complexes at the cell pole and the lateral cell wall). Real-time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain pmf under stress conditions. The two spatial subspecies therefore appear to be dynamically and functionally distinct with the polar clusters being associated with sensory function and the mobile complexes with maintenance of pmf.

Diacylglycerol and protein kinase D localization during T lymphocyte activation.

The serine kinase protein kinase D (PKD) has a cysteine-rich domain (CRD) that binds diacylglycerol (DAG) with high affinity. PKD is cytosolic in unstimulated T cells, but it rapidly polarizes to the immunological synapse in response to antigen/antigen presenting cells (APCs). PKD repositioning is determined by the accumulation of DAG at the immunological synapse and changes in DAG accessibility of the PKD-CRD. Unstimulated T cells are shown to have a uniform distribution of DAG at the plasma membrane, whereas after T cell activation, a gradient of DAG is created with a persistent focus of DAG at the center of the synapse. PKD is only transiently associated with the immune synapse, indicating a fine tuning of PKD responsiveness to DAG by additional regulatory mechanisms. These results reveal the immune synapse as a focal point for DAG and PKD as an immediate and dynamic DAG effector during T cell activation.

Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.

Protein kinase C and beyond.

Protein kinase C molecules regulate both positive and negative signal transduction pathways essential for the initiation and homeostasis of immune responses. There are multiple isoforms of protein kinase C that are activated differently by calcium and diacylglycerol, and these are activated mainly by antigen receptors in T cells, B cells and mast cells. Additionally, mammals express several other diacylglycerol binding proteins that are linked to a network of key signal transduction pathways that control lymphocyte biology. Diacylglycerol and protein kinase C regulate a broad range of gene transcription programs but also modulate integrins, chemokine responses and antigen receptors, thereby regulating lymphocyte adhesion, migration, differentiation and proliferation.