Paolo Ronchi

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

Summary Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the ‘Golgi-depleted’ karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

Abstract Laser-mediated nanosurgery has become popular in the last decade because of the previously unexplored possibility of ablating biological material inside living cells with sub-micrometer precision. A number of publications have shown the potential applications of this technique, ranging from the dissection of sub-cellular structures to surgical ablations of whole cells or tissues in model systems such as Drosophila melanogaster or Danio rerio. In parallel, the recent development of micropatterning techniques has given cell biologists the possibility to shape cells and reproducibly organize the intracellular space. The integration of these two techniques has only recently started yet their combination has proven to be very interesting. The aim of this review is to present recent applications of laser nanosurgery in cell biology and to discuss the possible developments of this approach, particularly in combination with micropattern-mediated endomembrane organization.

Positive feedback between Golgi membranes, microtubules and ER exit sites directs de novo biogenesis of the Golgi

ABSTRACT The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question, we have used laser nanosurgery to deplete BSC1 cells of the Golgi complex and have monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, endoplasmic reticulum (ER) export is inhibited and the number of ER exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers by microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Taken together, on the basis of our results, we propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive-feedback loops.

Single cell polarity in liquid phase facilitates tumour metastasis

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.Polarisation of metastasising cancer cells in circulation has not been investigated before. Here the authors identify single cell polarity as a distinct polarisation state of single cells in liquid phase, and show that perturbing single cell polarity affects attachment, adhesion, transmigration and metastasis in vitro and in vivo.

Golgi depletion from living cells with laser nanosurgery.

How Golgi biogenesis occurs in mammalian cells is a controversial problem. Can the Golgi complex (GC) form de novo from ER membranes or does it require a template? The method described in this chapter uses growth of cells on micropatterns to displace the GC from its juxtanuclear position and laser nanosurgery to subsequently deplete it from living cells. Golgi-depleted karyoplasts can be followed by time-lapse microscopy to address if and how the GC can be de novo synthesized from ER membranes. Furthermore, the study of different processes in the absence of the GC can shed light on the role of this organelle in the intracellular signaling and homeostasis.

Tunneling nanotubes contribute to the stroma-mediated imatinib resistance of leukemic cells

Intercellular communication within the bone marrow niche significantly influences leukemogenesis and the sensitivity of leukemic cells to therapy. Tunneling nanotubes (TNTs) are a novel mode of intercellular cross-talk. They are long, thin membranous protrusions that enable the direct transfer of various cargo between cells. Here we show that TNTs are formed between leukemic and bone marrow stromal cells. Fluorescence confocal microscopy with 3D reconstructions, correlative light-electron microscopy and electron tomography provided evidence that TNTs transfer cellular vesicles between cells. The quantitative analysis demonstrated that the stromal cells stimulate TNT-mediated vesicle transfer towards leukemic cells. Transfer of vesicular cargo from stromal cells correlated with increased resistance to anti-leukemic treatment. Moreover, specific sets of proteins with a potential role in survival and the drug response were transferred within these vesicles. Altogether, we found that TNTs are involved in the leukemia-stroma cross-talk and the stroma-mediated cytoprotection of leukemic cells. Our findings implicate TNT connections as a possible target for therapeutic interventions within the leukemia microenvironment to attenuate stroma-conferred protection.

Alpha-synuclein fibrils induce autophagy in microglial cells as a consequence of lysosomal damage.

Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. In Parkinson’s disease, the synaptic protein alpha-synuclein (AS) accumulates in neuronal cell bodies and axons. Recent studies indicate that aggregation-prone proteins can spread to other brain cells - such as glia - contributing to progressive deterioration. Although autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, exact mechanisms are not clear and most work was done in neurons and not on microglial cells. Here we report that AS fibrils but not monomers induce lysosomal damage and autophagy in microglial cells and we extensively characterized the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM). In addition, we found that autophagy inhibition in these cells impairs mitochondrial quality and leads to microglial cell death. We propose that AS accumulation in lysosomes leads to lysosomal damage, which in turn activates canonical autophagy as a rescue mechanism. Our results provide novel findings about the interaction between AS and the autophagy pathway in microglial cells, which may be important for targeting protein misfolding-associated neurodegenerative diseases.

Targeted Ablation Using Laser Nanosurgery

Laser-mediated dissection methods have been used for many years to micro-irradiate biological samples, but recent technological progress has rendered this technique more precise, powerful, and easy to use. Today pulsed lasers can be operated with diffraction limited, sub-micrometer precision to ablate intracellular structures. Here, we discuss laser nanosurgery setups and the instrumentation in our laboratory. We describe how to use this technique to ablate cytoskeletal elements in living cells. We also show how this technique can be used in multicellular organisms, to micropuncture and/or ablate cells of interest and finally how to monitor a successful laser nanosurgery.