Rosella Visintin

Rosella Visintin Lecture

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Rosella Visintin

Abstract

Rosella Visintin


Clb2 phosphorylation and de-phosphorylation dynamics set   the conditions for anaphase  progression

Mitotic events are orchestrated by oscillations in the activity of cyclin-dependent kinase 1 (Cdk1), which is modulated by the mitotic cyclins (Clb2 in yeast) associating with it. To progress through anaphase and next exit from mitosis, cells must inactivate Cdk1. The latter is achieved mainly via proteolysis of Clb2 by the proteasome. Clb2 degradation is mediated by the ubiquitin ligase APC/C (anaphase-promoting complex) in association with co-factor Cdc20 or Cdh1. Clb2 degradation is bi-phasic; about half of Clb2 molecules is turned over at the metaphase-anaphase transition by the APC/C-Cdc20, while the rest becomes degraded at mitotic exit by the APC/C-Cdh1.

One question that has remained unanswered is why and how APC/C-Cdc20 degrades only half of the Clb2 pool. We propose that Cdc5-mediated phosphorylation of Clb2 represents a signal for APC/C-Cdc20 recognition. Concomitantly with Clb2 turn over, the phosphatase Cdc14 becomes activated. This initial activation of Cdc14 requires the function of Clb2. We show that a non-phosphorylatable Clb2 protein besides being protected from degradation is also impaired in Cdc14 release. Taken together, Clb2 phos-phorylation by Cdc5 is required for APC/C-Cdc20-mediated Clb2 degradation and for Clb2 function within the FEAR network to promote Cdc14 activation. Active Cdc14 dephosphorylates Clb2 therewith restricting its own activity at early anaphase and protecting the remaining Clb2 molecules from degrading.

Vincenzo-Costanzo

Vincenzo Costanzo Lecture

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Vincenzo Costanzo

Abstract





Mechanisms of replication fork protection

Maintenance of genome integrity during DNA replication relies on several factors that do not directly participate in DNA synthesis. Among these there are proteins that respond to DNA lesions and structures impairing replication fork progression. These factors are believed to be particularly important for the resolution of DNA damage occurring during DNA replication and the faithful duplication of chromosome regions with complex or repetitive DNA sequences. In my group, we combine the power of the Xenopus egg extract with advanced imaging techniques based on transmission electron microscopy (EM) to study major DNA metabolism processes linked to DNA replication and DNA repair. This approach allowed us to uncover for the first time a role for RAD51 in protecting replication forks, by preventing Mre11 mediated processing of nascent DNA (Hashimoto et al 2010, NSMB). These findings have been confirmed in mammalian cells by several other studies and linked to cancer cell survival and sensitivity to chemotherapy. More recently, we have shown that extensive nascent DNA degradation is triggered by the formation of reversed forks at stalled replication intermediates operated by SNF2 helicases (Kolinjivadi et al 2017, Mol Cell). These pathways are likely to play a major role in the presence of DNA damage and at complex DNA loci, including the centromeres, which we have shown to be enriched for DNA repair factors (Aze et al 2016, NCB). This work will be discussed in light of recent results obtained on replication fork protection mechanisms.


Federico Forneris Lecture

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Federico Forneris

Abstract





Molecular architectures, interactions and functions of neuromuscular synapse  organizers

Synapse formation is a finely-tuned process requiring multiple steps and a variety of receptors and signaling molecules. The neuromuscular synapse or junction (NMJ) – the connection between motor neurons and skeletal muscles – represents the archetype junction system for studying synapse formation and conservation. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the construction of a highly specialized postsynaptic architecture juxtaposed to a highly differentiated nerve terminal. Our research focuses on the investigation of the key signaling molecules and receptors that enable NMJ formation and stabilization.

We study the extracellular architecture and function of two subsets of proteins tied to the role of agrin, a well known NMJ stabilizer. The first subset includes the agrin receptor, comprising the  receptor tyrosine kinase MuSK (muscle-specific kinase) and its co-receptor LRP4; the second includes the soluble synapse-specific regulatory protease Neurotrypsin (NT) that cleaves agrin with high specificity. This focus has yielded novel insight into processes underlying NMJ formation. In the first case, crystal structures of MuSK ectodomain fragments, coupled with solution scattering data and cross-linking mass spectrometry, highlighted the contribution of molecular flexibility in receptor activation and signaling. In the second case, in vitro enzyme studies and cell-based assays elucidated the specificity of NT, revealing a delicate regulatory mechanism mediated by splicing variants, ion binding, and post-translational modifications. Our results offer new understanding of NMJ formation and stabilization, and contribute innovative tools to the characterization of this  fundamental biological system.

Iannacone

Matteo Iannacone Lecture

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Matteo Iannacone

Abstract


Iannacone




Spatiotemporal dynamics and genomic landscape of CD8+ T cells undergoing intrahepatic priming

CD8+ T cell responses to hepatotropic viruses like HBV range from dysfunction to differentiation into effector cells, but the mechanisms underlying these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells –not natural targets of HBV – leads to differentiation into effector cells that form dense, extravascular clusters of rather immotile cells scattered throughout the liver. By contrast, priming by hepatocytes – natural targets of HBV – leads to local activation and proliferation but lack of differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses unveil unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by anti-PD-L1 treatment, but instead respond to IL-2. These findings suggest new immunotherapeutic strategies against chronic HBV infection.