Research Interests

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The genome of eukaryotic cells is compartmentalized inside the nucleus and separated from the cytoplasm allowing a level of regulation which is unprecedented in prokaryotes. This separation is achieved by the nuclear envelope, which is formed by two membranes, the inner and the outer nuclear membrane. Embedded in this two-membrane structure are nuclear pore complexes, the gatekeepers of the nucleus. These are huge macromolecular assemblies of around 60 MDa in vertebrates which allow transport between the cytoplasm and the nucleoplasm during interphase.

 
Schematic drawing of the nucleus and the nuclear envelope
 

In interphase, the chromatin (blue) of the nucleus is surrounded by the nuclear envelope that consists of two membranes in which nuclear pore complexes (red) are embedded: The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum whereas the inner nuclear membrane has a unique protein composition.

 
In higher eukaryotes, the nuclear envelope is a highly dynamic structure during the cell cycle. Whereas yeast cells segregate the chromatin during mitosis with the nuclear envelope remaining intact – a so called closed mitosis – in animal cells, the nuclear envelope breaks down at the beginning of mitosis. This means that nuclear pore complexes are disassembled into mostly soluble proteins or protein sub-complexes, which are dispersed all over the cytoplasm. The membranes of the nuclear envelope are, at least in mammals, reabsorbed into the endoplasmic reticulum. At the end of mitosis two new nuclear envelopes reform around the separated chromatin, i.e. the two-membrane structure and nuclear pore complexes re-assemble. This process is tightly coordinated and regulated: Although nuclear pore complexes are assembled from mostly cytosolic components, for assembly they need the presence of membranes in which they are integrated. On the other hand, formation of the two membrane structure without nuclear pore complexes would be disastrous for the cell, as the communication and transport between cyto- and nucleoplasm would be blocked.

The metazoan nucleus undergoes a dramatic structural and functional changes during the cell cycle

Chromatin (blue) condenses during entry into mitosis and the nuclear envelope breaks down so that the mitotic spindle can form. After chromosome segregation in anaphase, the nuclear envelope including pore complexes reforms in telophase. Nuclear pore complexes or precursors are shown in red, microtubules in green.


We are interested in the molecular mechanisms of nuclear envelope and nuclear pore complex formation as well as chromatin decondensation. Using a cell free system based on cellular extracts prepared from Xenopus laevis eggs we can reconstitute these processes in a test tube and dissect them biochemically, identify the key components involved and define how they function.

 
 

 
Preparation of Xenopus laevis egg extracts

The upper panel shows the frogs in our aquarium system and the eggs they lay. Lower panels show the preparation of egg extracts: Eggs are crashed by centrifugation to obtain a low speed extract. This extract can be further separated into cytosolic and membrane fractions.

 
Upon incubation of the cytosol and a membrane fraction with sperm chromatin as a template, nuclei form in vitro. These nuclei contain a closed nuclear envelope and nuclear pore complexes that are able to import proteins. The incorporated DNA is faithfully replicated and the nuclei can be triggered to break down by artificially inducing the extracts to re-enter mitosis.
 


Nuclear envelope reformation in vitro


Nuclei form when any source of DNA (in this case frog sperm DNA) is incubated with cytosol and membranes derived from Xenopus laevis egg extracts. The upper panel shows the individual steps of the process: First, membranes bind on the DNA surface (2nd picture) and fuse to form a closed nuclear envelope (3rd picture) with the DNA getting massively decondensed. (4th picture).  Membranes are stained with a red fluorescent membrane dye (DiIC18), DNA with DAPI (blue). The lower panel shows an immunostaining for nuclear pore complexes which are embedded in the nuclear envelope (left) and electron microscopy pictures of a nucleus (middle) and magnifications of the nuclear envelope showing two nuclear membranes and nuclear pore complexes in the centre). Scale bars are 10µm, for the electron microscopy magnifications of the nuclear envelope 100nm.

 
The nuclear envelope reforms around the segregating and decondensing chromatin. Little is known about chromatin decondensation during mitotic exit, the individual steps and factors involved and how it is regulated and coordinated with other concomitant cellular processes.
 

Chromatin decondensation in vitro

Mitotic chromatin (isolated as clusters from tissue culture cells) are incubated with Xenopus egg extracts to induce chromatin decondensation. At the end of the reaction an interphasic nucleus is formed. DNA is stained with DAPI, bar is 5µm.