RuvBL1/2 function in mitotic chromatin decondensation

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The nucleus reorganizes structurally and functionally considerably during mitosis. In animal cells, the nuclear envelope breaks down at the beginning of mitosis, the chromatin massively condenses to individualized chromosomes which are captured and segregated by the spindle apparatus - processes which have been and still are intensively studied in many laboratories. Much less is known how at the end of mitosis the interphase state of the nucleus is reestablished, competent for its manifold functions. For this, the highly condensed mitotic chromosomes are decompacted, a process indispensable for reinitiating transcription and the perpetuation of genomic information and thus of central importance in the cellular life cycle. Despite its significance to basic research as well as its potential medical implications, mitotic chromatin decondensation is still ill-defined. It has been well and early described cytologically, but we are largely ignorant of the underlying molecular events and mechanisms.We have previously identified and characterized the ATPase complex RuvBL1/2 as a crucial chromatin decondensation factor. By SILAC based mass spectrometry from cells exiting mitosis we now identify eIF4A1, PKP1 and JUP as RuvBL1/2 interactors, which bind specifically during the time of chromatin decondensation. Indeed, downregulation or depletion of the proteins in cells or in biochemical assays impairs chromatin decondensation. eIF4A1 is a RNA helicase with a function in translation initiation but we propose an additional role in chromatin decondensation independent from its role in protein synthesis. PKP1 and JUP are desmosome components with an additional localization in the nucleus but their precise function in this compartment is largely unclear. Here, we will analyze how eIF4A1, PKP1 and JUP precisely function in chromatin decondensation using cellular assays including life cell imaging and biochemical methods like a cell free reconstitution assay of mitotic chromatin decondensation. We will determine whether eIF4A1, PKP1 and JUP form a single chromatin decondensation complex involving RuvBL1/2 or whether they are part of different RuvBL1/2 containing complexes mediating distinguishable aspects of chromatin decondensation. We will analyze whether the chromatin decondensation complex(es) localize to chromatin during mitotic exit similar to RuvBL1/2 and if so determine which components of the complex(es) define chromatin binding. By depleting the crucial decondensation components in tissue culture cells and the in vitro decondensation assay we will analyze which aspects of mitotic exit (chromatin compaction, nuclear envelope reformation, reassembly of subnuclear structures etc.) are affected and determine how the involved proteins act in the processes. We will shed light on an ill-defined but important cell biological process at the end of mitosis indispensable to reestablish nuclear structure and function.