Tilo Wolf Eichler

The role of myosin Va in secretory granule trafficking and exocytosis

It emerges that myosin Va plays multiple roles in the trafficking of SGs (secretory granules). In addition to a function in the capture and transport of newly formed SGs in the F-actin-rich cortex, myosin Va is implicated in late transport events of these organelles, which precede their exocytosis. Consistent with these roles, interactions of myosin Va with an array of well-known proteins involved in regulated protein secretion have been documented.

A Unified Framework for Automated 3-D Segmentation of Surface-Stained Living Cells and a Comprehensive Segmentation Evaluation

This work presents a unified framework for whole cell segmentation of surface stained living cells from 3-D data sets of fluorescent images. Every step of the process is described, image acquisition, prefiltering, ridge enhancement, cell segmentation, and a segmentation evaluation. The segmentation results from two different automated approaches for segmentation are compared to manual segmentation of the same data using a rigorous evaluation scheme. This revealed that combination of the respective cell types with the most suitable microscopy method resulted in high success rates up to 97%. The described approach permits to automatically perform a statistical analysis of various parameters from living cells.

Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

CDR2 antigen and Yo antibodies.

Paraneoplastic cerebellar degeneration (PCD) is often associated with Yo antibodies that are directed against human cerebellar degeneration-related protein 2 (CDR2). Such antibodies may also be found in ovarian cancer patients without PCD. We studied if there was an association between Yo antibody production and differences in CDR2 cDNA sequence, mRNA or CDR2 expression in ovarian cancers. We found similar CDR2 cDNA sequence, mRNA and protein levels in primary ovarian cancers, with or without associated Yo antibodies. CDR2 was also present in other cancers, as well as in normal ovary tissue. The results suggest that Yo antibodies are not only related to the expression of CDR2 alone, but also to immune dysregulation.

CDR2L Antibodies: A New Player in Paraneoplastic Cerebellar Degeneration

Objective Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens. Methods Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins. Results RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane. Interpretation Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD.

Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

A novel nanoprobe for multimodal imaging is effectively incorporated into human melanoma metastatic cell lines

To facilitate efficient drug delivery to tumor tissue, several nanomaterials have been designed, with combined diagnostic and therapeutic properties. In this work, we carried out fundamental in vitro and in vivo experiments to assess the labeling efficacy of our novel theranostic nanoprobe, consisting of glycogen conjugated with a red fluorescent probe and gadolinium. Microscopy and resazurin viability assays were used to study cell labeling and cell viability in human metastatic melanoma cell lines. Fluorescence lifetime correlation spectroscopy (FLCS) was done to investigate nanoprobe stability. Magnetic resonance imaging (MRI) was performed to study T1 relaxivity in vitro, and contrast enhancement in a subcutaneous in vivo tumor model. Efficient cell labeling was demonstrated, while cell viability, cell migration, and cell growth was not affected. FLCS showed that the nanoprobe did not degrade in blood plasma. MRI demonstrated that down to 750 cells/μL of labeled cells in agar phantoms could be detected. In vivo MRI showed that contrast enhancement in tumors was comparable between Omniscan contrast agent and the nanoprobe. In conclusion, we demonstrate for the first time that a non-toxic glycogen-based nanoprobe may effectively visualize tumor cells and tissue, and, in future experiments, we will investigate its therapeutic potential by conjugating therapeutic compounds to the nanoprobe.

Paraneoplastic syndrome-associated neuronal antibodies in adult ADHD

A high seroprevalence of Yo antibodies targeting cerebellar Purkinje cells was recently reported in children with attention deficit/hyperactivity disorder (ADHD). We investigated the presence of 8 paraneoplastic neurological syndrome (PNS)-associated antibodies including anti-Yo in 169 adult ADHD patients. No associations between ADHD and serum Yo antibodies or other antibodies associated with PNS were found. However, 10 out of 48 ADHD patient sera analyzed by immunofluorescence presented antibodies targeting cerebellar Purkinje cells. This reactivity probably represents the presence of low levels of antibodies against multiple cellular hitherto unknown antigens with little to no clinical significance.

CCDC104 Antibodies and Mitosis of Cancer Cells

Dear Editor, We have previously identified cancer patients with the paraneoplastic antibody, anti-Yo and the less characterized antibody, anti-CCDC104 (coiled-coil domain containing protein 104, also referred to as CFAP36 or BARTL1) [1]. CCDC104 has been localized to both the nucleus and cytoplasm of neuroblastoma cells [1], and the protein may have a role in the formation of cellular cilia [2, 3]. Anti-Yo consists of antibodies against cerebellar degeneration-related protein 2 (CDR2) and cerebellar degeneration-related protein 2 like (CDR2L), respectively [4, 5]. CCDC104, CDR2 and CDR2L have been found in normal brain and cancer tissue [1], but whether these proteins are involved in related cell functions remains unknown. Sera from a post-menopausal woman with fallopian tube cancer and a paraneoplastic cerebellar degeneration were tested at different time points of the disease. Serum antibodies against CDR2/CDRL (anti-Yo) were identified by immunohistochemistry, radioimmunoassay and line blot, whereas CCDC104 antibodies were identified by radioimmunoassay [1]. To localize the cellular binding of CCDC104 antibodies, we stained HeLa (ATCC-CCL-2) cells grown on poly-L-Lysine-coated 10-mm glass coverslips with the patient serum (diluted 1:200 in 0.2% gelatin in PBS) and a polyclonal antibody against CCDC104 (Sigma HPA008994 diluted 1:200). Secondary antibodies coupled to Alexa Fluor (AF) 488 or 594 (Invitrogen antirabbit-AF594 A11012 and anti-human-AF488 A11013) were used at 1:300 dilutions. Coverslips were mounted with ProLong Gold/DAPI (Invitrogen, P36931), and imaging was performed with a Zeiss Axiovert M200 fluorescence microscope with a 409 objective and TILL photonics monochromator. We found that Yo and CCDC104 antibodies coexisted in the same patient and that CCDC104 antibodies were localized to the spindle apparatus in dividing HeLa cells (Fig. 1A,B). Furthermore, the CDR2, CDR2L and CCDC104 antibody indexes determined by radioimmunoassay were stable for more than ten years after cancer treatment (Fig. 1C). Our results suggest that CCDC104 plays a role in mitotic cell division. CDR2 has also been linked to cell mitosis [6] although neither CDR2 nor CDR2L appear to localize to the spindle apparatus. Further, we have shown that CDR2 and CDR2L antibodies localize to the cytoplasm and that CDR2L antibodies also bind to the cell membrane of HeLa cells, but their exact organelle localization has yet to be determined [4]. We have shown that CDR2/CDR2L antibodies play an important role in paraneoplastic cerebellar degeneration by inducing Purkinje neuron death [5]. It is unlikely that CCDC104 antibodies contribute to this process as the CCDC104 protein appears to have role in cell mitosis, which does not occur in Purkinje neurons. While the 2011 2004 200

Abstract 5195: A novel, multimodal theranostic nanoprobe is effectively incorporated into melanoma brain metastatic cells

Metastatic cancer is a significant cause of death worldwide, despite continuing advances in research on diagnostics and therapy. To overcome unsuccessful drug delivery to the tumor tissue, several different nanoprobes have been designed, of which some have combined diagnostic and therapeutic properties. It is an advantage to use non-toxic, biodegradable materials in the design of nanoprobes, as such compounds are easily eliminated from the body. In this work we report for the first time the use of a recently developed nanoprobe to label melanoma metastatic cell lines, for multimodal imaging of cellular uptake and drug delivery. Our nanoprobe consisted of a backbone of glycogen, where the red fluorescent marker Dyo-615 for fluorescence microscopy and Gadolinium for MRI were incorporated. Fluorescence microscopy showed effective labeling of human melanoma metastatic cells over 24 hours and cell viability was not affected by the labeling. The nanoprobe was found in the cytosol of the cells, and a gradual degradation of the probe inside the lysosomes could be observed. In vitro MRI relaxivity measurements showed significant reduction in T1 mapping times, compared to unlabeled cells. In vivo MRI studies showed that subcutaneous melanoma tumors in mice could be effectively visualized with T1 weighted MRI after intravenous injection of the nanoprobe, and the contrast enhancement was comparable to what was seen after using standard Omniscan contrast agent. In summary, our biodegradable glycogen nanoprobe shows a high potential to be used further as a theranostic entity. The nanoprobe may offer additional advantages over MRI contrast agents, as tumor uptake of pharmaceuticals attached to the nanoprobe can be traced in real-time in vivo. Citation Format: Synnove N. Aasen, Tilo W. Eichler, Martin Hruby, Aneta Pospisilova, Petr Stepanek, Endy Spriet, Daniel Jirak, Kai Ove Skaftnesmo, Frits Thorsen. A novel, multimodal theranostic nanoprobe is effectively incorporated into melanoma brain metastatic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2015-5195