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Nuclear structure, function and dynamics

The defining compartment of a eukaryotic cell is the cell nucleus, in which the genetic information is stored in form of chromatin. The chromatin is enwrapped by the nuclear envelope (NE) that lies on top of the nuclear lamina and contains nuclear pore complexes (NPCs) to allow for the exchange of molecules between the nucleus and the cytoplasm.

Our lab is interested in two different aspects of nuclear biology.

First, we study the dynamics of the NE during cell division in higher eukaryotes. Disintegration of the NE upon entry into mitosis is a prerequisite for the proper segregation of genetic information during cell division in vertebrate cells. Nuclear envelope breakdown (NEBD) involves the disassembly of the NPCs, the depolymerization and solubilization of the nuclear lamina and detachment and removal of the nuclear membrane from the chromatin. We are investigating the molecular mechanisms of NEBD with a focus on the disassembly of NPCs.

Our second focus is to understand the highly regulated and mechanistically unique processes responsible for the production of ribosomal subunits in higher eukaryotes. Pre-ribosomal subunits are produced in a nuclear subdomain, the nucleolus, involving a series of RNA-processing, RNA-modifying and protein assembly steps. Then, pre-ribosomal subunits are released from the nucleolus into the nucleoplasm, where they undergo further maturation and are finally exported to the cytoplasm to function in protein biosynthesis. We are elucidating the role of trans-acting factors in the assembly pathway and study how nuclear and cytoplasmic maturation steps are coordinated.

(A) As cells enter mitosis, microtubules attaching to the outer face of NE tear the NE resulting in formation of 1 to 3 large holes. Concomitantly, nuclear lamina (green) and NPC disassembly starts involving CDK1/cyclinB and PKC-dependent phosphorylation of lamins and nucleoporins. Nuclear membrane proteins are retracted into the ER, where they reside in metaphase.
On the right, NPCs are visualized by immunostaining. The anti-Nup153 antibody decorates the nuclear rim in interphase cells (median) and gives rise to a dotty pattern on the nuclear surface. During mitosis, most nucleoporins are solubilized and released into the cytoplasm.

(B) Scheme illustrating the maturation of 40S and 60S subunits. Concomitant with rRNA transcription, modification of rRNA and ribosome assembly starts, leading to a pre-90S particle, which contains rRNA processing and modification factors as well as many ribosomal proteins of the small subunit (rpS proteins). rRNA cleavage liberates a pre-40S particle. Pre-60S particles are formed by binding of rpL proteins and accessory factors to the rRNAs of the large subunit. After further remodeling steps in the nucleoplasm, preribosomal subunits are exported to the cytoplasm, export factors are released and the subunits undergo final maturation steps.
HeLa cells were treated with Leptomycin B (LMB), a drug that inactivates the exportin CRM1 (right panel). Indirect immunofluorescence using antibodies to rpL23a and rpS6 reveals nuclear accumulation of both markers, indicating that export of both subunits depends on the CRM1 export pathway.

 
 
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Thu Apr 27 14:45:39 CEST 2017
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