A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

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The sorting nexins SNX1 and SNX2 are members of the retromer complex involved in protein sorting within the endocytic pathway. While retromer-dependent functions of SNX1 and SNX2 have been well documented, potential retromer-independent roles remain unclear. Here, we show that SNX1 and SNX2 interact with the Rac1 and RhoG guanine nucleotide exchange factor Kalirin-7. Simultaneous overexpression of SNX1 or SNX2 and Kalirin-7 in epithelial cells causes partial redistribution of both SNX isoforms to the plasma membrane, and results in RhoG-dependent lamellipodia formation that requires functional Phox homology (PX) and Bin/Amphiphysin/Rvs (BAR) domains of SNX, but is Rac1- and retromer-independent. Conversely, depletion of endogenous SNX1 or SNX2 inhibits Kalirin-7-mediated lamellipodia formation. Finally, we demonstrate that SNX1 and SNX2 interact directly with inactive RhoG, suggesting a novel role for these SNX proteins in recruiting an inactive Rho GTPase to its exchange factor.

A novel, retromer-independent role for sorting nexins 1 and 2 in RhoG-dependent membrane remodeling.

Recent studies suggest that extracellular matrix (ECM) components within the tumor microenvironment can influence malignant progression, thus we investigated the influence of the ECM binding receptor β1 integrin, on the hallmark properties of tumorigenesis. Small interfering (si) or short hairpin (sh) RNA approaches were used to deplete β1 integrin in cancer cell lines. β1 integrin-depleted cells were then assessed for their growth and invasive capabilities using 2-dimensional (2D) or 3D culture conditions. Depletion of β1 integrin expression did not impact cell growth in 2D assay systems; however, β1 integrin and its ligand fibronectin were required for growth in 3D. β1 integrin-depleted cells also had reduced invasive capabilities, in part due to increased tissue inhibitor of metalloprotease (TIMP)-2 expression in conjunction with down-regulation of matrix metalloprotease (MMP)-9 levels in β1 integrin-depleted cells. Our results suggest that despite no apparent effect on 2D cell growth, fibronectin-β1 integrin signaling is a critical mediator of the 3D growth and invasive properties of tumor cells. These observations highlight the importance of investigating the role of adhesion molecules in the appropriate context and furthermore identify β1 integrin as a possible therapeutic target to inhibit the aggressive growth and invasion of tumor cells.

β1 integrin is required for anchorage-independent growth and invasion of tumor cells in a context dependent manner.

A proline to serine mutation (P56S) in vesicle-associated membrane protein-associated protein B and C (VAPB) causes an autosomal dominant form of amyotrophic lateral sclerosis (ALS). We show that the mutation also causes a nuclear envelope defect. Transport of nucleoporins (Nups) and emerin (EMD) to the nuclear envelope is blocked, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanine residues in an acidic track) antagonizes the effect of mutant VAPB and restores transport to the nuclear envelope. VAPB function is required for transport to the nuclear envelope, with knockdown of endogenous VAPB recapitulating this phenotype. Moreover, we identified the compartment into which the Nups and EMD were sequestered as the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), with nuclear envelope membrane proteins transiting to the ERGIC before VAPB-dependent retrograde transport to the nuclear envelope.

A mutation in VAPB that causes amyotrophic lateral sclerosis also causes a nuclear envelope defect.

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C-terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly.

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation.

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation.

Xenopus egg extracts have been widely used to study cell cycle regulation and to analyze mitotic or nuclear processes on a biochemical level. Most instrumental, proteins of interest can be immunodepleted by specific antibodies. However, this approach has been restricted to non-membrane proteins, which limits its versatility especially when studying membrane-dependent processes such as nuclear envelope reformation at the end of mitosis or nuclear pore complex assembly. We describe here the methods developed and used in our laboratory to specifically remove transmembrane proteins from endogenous membranes and to insert recombinant integral membrane proteins into endogenous membranes. The latter procedure is important not only for readdition of a depleted protein in rescue experiments but also for introducing artificial membrane proteins such as reporters to investigate the passage of inner nuclear membrane proteins through nuclear pore complexes.

Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

RuvB-like ATPases function in chromatin decondensation at the end of mitosis.

In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.

Nup153 Recruits the Nup107-160 Complex to the Inner Nuclear Membrane for Interphasic Nuclear Pore Complex Assembly.

The metazoan nucleus breaks down and reassembles during each cell division. Upon mitotic exit, the successful reestablishment of an interphase nucleus requires the coordinated reorganization of chromatin and formation of a functional nuclear envelope. Here, we report that the histone demethylase LSD1 (also known as KDM1A) plays a crucial role in nuclear assembly at the end of mitosis. Downregulation of LSD1 in cells extends telophase and impairs nuclear pore complex assembly. In vitro, LSD1 demethylase activity is required for the recruitment of MEL28 (also known as ELYS and AHCTF1) and nuclear envelope precursor vesicles to chromatin, crucial steps in nuclear reassembly. Accordingly, the formation of a closed nuclear envelope and nuclear pore complex assembly are impaired upon depletion of LSD1 or inhibition of its activity. Our results identify histone demethylation by LSD1 as a new regulatory mechanism linking the chromatin state and nuclear envelope formation at the end of mitosis.

Allana Schooley

Friedrich Miescher Laboratory

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A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Allana Schooley

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max Planck society

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Friedrich Miescher Laboratory