2019 – Darshan Kumar, Banafsheh Golchoubian, Ilya Belevich, Eija Jokitalo, and Anne-Lore Schlaitz. REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis. Molecular Biology of the Cell.
2018 – Neeraj Prabhakar, Anni Määttänen, Jouko Peltonen, Pekka Hänninen, Markus Peurla, Jessica M Rosenholm. Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy. Microscopy (Oxf)
2018 – Prabhakar N, Peurla M, Koho S, Deguchi T, Näreoja T, Chang HC, Rosenholm JM, Hänninen P. STED-TEM Correlative Microscopy Leveraging Nanodiamonds as Intracellular Dual-Contrast Markers. Small
Method: Correlative Light and Electron Microscopy (CLEM)
Sorting of proteins for transport to the cells surface or for secretion from cells is essential for the extracellular activities of proteins like insulin, surface receptors, secreted proteases, and GPCRs to name a few. A major regulator of intracellular protein distribution is the trans-golgi network (TGN) that sorts secretory proteins into specific carriers to transport them to their final destination. The sorting of lysosomal hydrolases at the TGN by the mannose-6-phosphate receptor is well established. However, the sorting mechanism for secreted proteins remains poorly understood. In recent years we have identified major components required for secretory cargo sorting at the TGN. These components include F-actin, cofilin, the Ca2 ATPase SPCA1 at the cytosol and Ca2 and the Golgi resident protein Cab45 in the lumen of the TGN. Although Ca2 and Cab45 are known to play a role in sorting the mechanism remained unclear. We can now show that Cab45 changes its conformation and oligomerizes in a Ca2 dependent manner. These oligomers specifically recruit cargo molecules such as Lysozyme-C and cartilage oligomeric protein in vitro. Furthermore, super-resolution microscopy revealed that the organization of Cab45, SPCA1 and cargo occurs in specific domains of the TGN. We believe that Cab45 is a sorter of secretory proteins and may represent a unique way to export cargo independent of a bona fide cargo receptor at the TGN.
- Spatial distribution of Cab45 in the Golgi and transport vesicles
- Information about the vesicle morphology and identity
Methods: Correlative Light and Electron Microscopy (CLEM), Electron Microscopy (EM), and Stimulated Emission Depletion (STED) Microscopy
Correlative light and electron microscopy (CLEM) makes it possible to combine the advantages of the two methods: specific molecules can be labeled and studied with a fluorescence microscope in live or fixed cell samples, after which high-resolution structural information of specific regions of interest can be obtained with TEM2,3. However, the resolution mismatch of several orders of magnitude between TEM and traditional fluorescence microscopes has posed significant limitations to the usefulness of such correlative experiments.Super-resolution fluorescence microscopy methods have the potential of alleviating the current problem., as they enable fluorescence imaging at the same resolution-scale with TEM, and recently several CLEM methods leveraging these techniques have been introduced. Stimulated Emission Depletion (STED) is a super-resolution fluorescence microscopy technique, based on point- scanning confocal microscopy.STED microscope can have comparable resolution with an electron microscope, up to 6-30nm. It could thus potentially be a very suitable partner for TEM in CLEM applications, as it maintains all the advantages of traditional fluorescence microscopy techniques (even 3D imaging of live cells), while providing a resolution comparable to that of TEM.In our experiments a 2D STED system was used, with inferior axial resolution (600nm), when compared to thin (100nm) TEM sections. This caused some problems in the finding the same 3D section in STED and TEM, because in practice, in every STED image NDs from approximately six TEM sections are visible. This however is not a fundamental limitation of the CLEM method, but of the specific instrument that was available to us. Such problem could be avoided by using a 3D STED and 3D serial sectioning system, which can provide sub-100nm in all three dimensions.
- Combining 3D gatan system with STED super resolution microscopy techniques would enable effective automated correlation of cells. 3D imaging mode that would automatically runs high-resolution and high content electron microscopy. On a more detailed level, there are to our knowledge no reports yet describing 3D CLEM using nanodiamonds as unique dual probe, which would enable the correlation of 3D cell volume with two high-resolution techniques.
- We are intending to improve the developed method further for 3D correlation of STED and TEM. 3D STED-TEM (CLEM technique) also allows improvements of microscopic techniques towards better ultra-high spatial/molecular resolution and ultra-high sensitivity will provide a better understanding of the cell’s complex machinery in basic research.
Methods: Stimulated Emission Depletion (STED) Microscopy, Total Internal Reflection Fluorescence (TIRF) Microscopy, Spinning Disk Confocal Microscopy (SDCM)
The Ca2 -binding EF-hand domain containing protein 2 (EFHD2 or Swiprosin1) is an actin bundling protein involved in cell spreading, migration and modulation of lamellipodial dynamics. Using the invasive breast cancer cell line MDA-MB- 231 as a model, we have observed that EFHD2 colocalises with β1-integrin at the cell leading front. Using Proximity ligation assays and live cell imaging approaches, we have observed that EFHD2 interacts with endocytosed β1-integrins and Rab21-containing vesicles. Moreover, knocking down EFHD2 affects β1-integrin endocytosis negatively and reduces the average speed of Rab21-containing vesicles. This process is dependent on the actin cytoskeleton. Thus, we would like to study how F-actin interacts with the integrin vesicles in the cell. We also want to investigate when and where do EFHD2, active β1-integrins and Rab21 interact during the endocytic process. Finally, we would like to observe the re-distribution of endocytosed β1-integrins in cells lacking EFHD2. Cells with depleted levels of EFHD2 show decreased migratory potential measured with a scratch wound assay, as well as increased adhesion to extracellular matrix components laminin-1, collagen and fibronectin. This suggests that the lack of EFHD2 increases the stability of integrin adhesions resulting in defective migration. Hence, we have hypothesised that expression of EFHD2 in breast cancer cells might enhance the turnover of integrin adhesion allowing the cells to efficiently migrate, increasing their invasive potential. Thus, elucidating the mechanism by which EFHD2 influences integrin transport will shed light on whether EFHD2 could be a potential therapeutic target for metastatic breast cancer.
- To observe and identify the actin structures formed around integrin vesicles
- To establish a methodology to image the endocytosis of beta1-integrins with a defined start point (temperature change)
- To observe how GFP-Rab21, mcherry-EFHD2 and active beta1-integrins interact during endocytosis
- To analyse whether the integrin vesicles are re-distributed to a specific endosomal compartment in cells lacking EFHD2 growing on micropatterns
Methods: Electron Microscopy (EM)
EphrinB2 and its tyrosine kinase receptor EphB4 are required for lymphatic vessel remodeling and valve formation during embryonic and early postnatal development. We sought to further analyze the cellular function of ephrinB2/EphB4 signaling in the lymphatic vasculature. Towards this aim, we employed conditional Efnb2 and Ephb4 mouse models in combination with lymphatic-specific CRE lines to induce loss of protein expression selectively in lymphatic endothelial cells at postnatal stages. Confocal microcopy analyses of dermal lymphatic vascualture revealed changes in cellular junctions and organisation upon loss of ephrinB2 or EphB4.
Electron microscopy analysis is expected to reveal defects not visible in light microscopy resolution therefore providing new insights into cellular mechanisms underlying lymphatic vascular defects in the ephrinB2/EphB4 mutant mice.
Methods: Stimulated Emission Depletion (STED) Microscopy, Spinning Disk Confocal Microscopy (SDCM)
Intermediate filament (IF) proteins form dynamic cytoskeletal array that do not only provide structural reinforcement to cells but in addition have a wide range of cellular functions in processes like cell migration and proliferation, cell organelle shaping and positioning, or the regulation of signalling molecules. There are over 70 different IFs expressed in a tissue-specific manner. The IF protein vimentin is found in cells of mesenchymal origin (Aebi et al. 2007). Even though vimentin KO mice have a rather mild phenotype, they show impaired or delayed wound healing. It has become clear that vimentin is involved in lamellipodia formation and integrin recycling. Previous studies have shown that vimentin deficient fibroblasts show impaired mechanical stability, randomized migration and delayed wound healing in scratch wound assays as well as disorganized and random orientation of focal adhesions during migration (Krieg et al. 1998). Vimentin is involved in formation of focal adhesions by regulating the Focal adhesion kinase (FAK) through activation of VAV2 (Guanine Exchange Factor). Additionally, vimentin interacts with actomyosin transverse arcs through linker protein plectin and controls the retrograde flow of actin. (Lappalainen et al. 2015)
Like any other Similar to other IFs, vimentin’s functions, mainly it’s assembly and disassembly, are controlled by phosphorylation. Significant in vivo phosphorylation sites have already been mapped out and their physiological functions are being studied. This project focuses on the influence of phosphosites of vimentin on cell migration as the underlying mechanism of this vital process are not fully understood yet. (Eriksson et al., 2007). The phosphosites which will be focused on are Ser-4,6,7,8,9 ; Ser- 38, Ser- 50, Ser- 55, Ser- 71,72 since those phosphorylation sites have been reported to be involved in different cellular functions. (Hyder et al. 2008)
- Show differences in organisation or alignment of focal adhesions during migration in a vimentin-deficient cell and vimentin-phosphomutant expressing cell.
- Insights into interaction between vimentin, actin arcs and focal adhesions through super resolution images.
Origin: United Kingdom of Great Britain and Northern Ireland
Methods: Laser Scanning Confocal Microscopy (LSCM), Optical Projection Tomography (OPT)
To identify Transcription Factors (TFs) that play a role in the Malpighian Tubules of Drosophila melanogaster (the equivalent of human kidney and liver), we used the expression resource, flyatlas.org, to identify a dozen TFs with strongly enriched (> 5x) expression. While some of these are already known, many are unknown in the context of renal development or function. The aim is to study these unknown TFs in this versatile animal model at the molecular and physiological level. TFs associated with abnormal development or morphology and/or abnormal fluid/ion transport will be investigated in mammalian systems by planned internships. The expected results are the identification of novel candidate genes including TFs involved with kidney ontogenesis.
The main goal is to obtain a clear picture of where the Malpighian Tubules are located within the fly (comparing control flies with mutant flies) without the necessity of any dissection, and having real 3D images for the first time in the literature. We expect to obtain informative images of where the tubules attach in our mutant conditions compared to the controls.
Methods: Laser Scanning Confocal Microscopy (LSCM), Selective Plane Illumination Microscopy (SPIM), Optical Projection Tomography (OPT)
Embryonic kidney development is characterized by a nephron patterning process which requires further understanding. Both chemical and mechanical factors play a role in this development. I plan to develop computational models of nephron patterning, which will require validation by ex vivo morphogenetic information using data obtained by bioimaging.
The project will provide ex vivo confirmation of the in silico model of early nephron patterning by allowing analysis of the spatiotemporal changes of cell aggregates both under free-running conditions and following targeted genetic manipulation. Hence, the project will be key to the success of my overall research, which primarily aims to advance our understanding of mammalian nephrogenesis.
- Description at cellular level of the morphogenetic processes during early kidney development
- Identification of key chemical effectors driving nephron development
- Identification of mechanical forces involved in early kidney development such as cell surface tensions related to cell proliferation and hypertrophy
Method: Electron Microscopy
We previously described REEP3 and REEP4 as microtubule-dependent remodelers of the endoplasmic reticulum (ER) in mitosis (Schlaitz et al., 2013). After loss of REEP3/4, ER associates with metaphase chromosomes. REEP3 and REEP4 have domains inducing high membrane curvature, we therefore speculate, that their loss may lead to more cisternal ER which can invade the spindle area and interfere with mitotic progression. Moreover, REEP3/4 may be involved in reshaping the nuclear envelope in early mitosis.
The EM project is highly important for our research as it will allow us to gain insight into the mechanisms of function of REEP3/4 in mitosis as well as to understand the roles of REEP proteins at the nuclear envelope. We expect answers to the following questions:
- Does depletion of REEP3/4 lead to a more cisternal ER in metaphase
- How is the ER distributed in a mitotic cell in wild type versus REEP3/4 knockdown?
- Are REEP3/4 involved in reshaping the nuclear envelope for nuclear envelope breakdown in prometaphase?
Methods: Electron Microscopy
Raptorial birds have the highest measured visual acuity. To achieve this ability raptorial birds have large eyes for their body size and a high acuity zone in their retina, the central fovea. In the central fovea, much of the retinal tissue is displaced creating a visible depression or pit in the retina. In this pit the photoreceptor cells are packed very tight and the width of each photoreceptor cell is very narrow. To understand the optical properties of these cells a detailed description of their geometry and their intracellular structures is required.
This project will provide critical data to understand how the visual acuity of raptors is achieved, together with the optical modeling of the photoreceptor cells we will understand the optical properties of these photoreceptors cells are. This will furthermore allow us to understand the limits of photoreceptor structure, how narrow can a photoreceptor cell be made before it fails in its light guiding properties?
Method: Selective Plane Illumination Microscopy (SPIM)
Abstract will be revealed after project completion.
Method: Electron Microscopy
Abstract will be revealed after project completion.