Merja Joensuu

Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells

During mitosis, ER network reorganization can lead to packing of the ER into tight concentric layers at the cell cortex and occurs in tandem with rounding of the cell. Morphometric and 3D EM analysis shows that in addition to reorganization, ER sheets undergo transformation toward more fenestrated and tubular forms before anaphase in mammalian cells.

Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets.

Understanding the structure–function relationship of cells and organelles in their natural context requires multidimensional imaging. As techniques for multimodal 3-D imaging have become more accessible, effective processing, visualization, and analysis of large datasets are posing a bottleneck for the workflow. Here, we present a new software package for high-performance segmentation and image processing of multidimensional datasets that improves and facilitates the full utilization and quantitative analysis of acquired data, which is freely available from a dedicated website. The open-source environment enables modification and insertion of new plug-ins to customize the program for specific needs. We provide practical examples of program features used for processing, segmentation and analysis of light and electron microscopy datasets, and detailed tutorials to enable users to rapidly and thoroughly learn how to use the program.

ER sheet persistence is coupled to myosin 1c –regulated dynamic actin filament arrays

Dynamic actin filament arrays localize to polygonal spaces between ER sheets and tubules and regulate lateral movement and transformations of sheets, that is, sheet persistence, and thereby the characteristic ER network architecture in cultured mammalian cells. Myosin 1c has a role in creating and/or maintaining ER-associated actin arrays.

Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

ABSTRACT Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. Summary: A genome-wide RNAi screen is used to identify several conserved regulators of nuclear actin import and polymerization, and thus links control of nuclear actin to many cellular processes.

Dendritic spine actin cytoskeleton in autism spectrum disorder

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the shape and size of dendritic spines correlate with the functional changes in excitatory synapses and are heavily dependent on the remodeling of the underlying actin cytoskeleton. Recent evidence implicates synapses at dendritic spines as important substrates of pathogenesis in neuropsychiatric disorders, including autism spectrum disorder (ASD). Although synaptic perturbations are not the only alterations relevant for these diseases, understanding the molecular underpinnings of the spine and synapse pathology may provide insight into their etiologies and could reveal new drug targets. In this review, we will discuss recent findings of defective actin regulation in dendritic spines associated with ASD.

Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles

Joensuu et al. describe a tool for subdiffractional tracking of internalized molecules. They reveal that synaptic vesicles exhibit stochastic switching between heterogeneous diffusive and transport states in live hippocampal nerve terminals.

NOGO-A/RTN4A and NOGO-B/RTN4B are simultaneously expressed in epithelial, fibroblast and neuronal cells and maintain ER morphology

Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.

ER sheet–tubule balance is regulated by an array of actin filaments and microtubules

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ER consist of dynamic and static subdomains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 ER, actin cytoskeleton and microtubules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 ER sheet transformations are coupled to a subset of dynamic actin filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Proteins involved in ER–actin cytoskeleton interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Myo1c regulation of actin arrays is conveyed to sheet morphology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Dynamic microtubules and actin filament arrays counterbalance sheet–tubule balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules

Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.