Nuclear Structure and Function

171

The nuclear pore protein Nup98 plays a conserved role in transcriptional memory. W. Light1, A. Thompson1, J. Freaney1, C. Horvath1, J. H. Brickner1; 1Molecular Biosciences, Northwestern University, Evanston, IL

The interaction of nuclear pore proteins (Nups) with genes can function to regulate transcription. In yeast, targeting of genes to the nuclear pore complex (NPC) promotes stronger transcription. For some inducible genes, interaction with the NPC is maintained for several generations after repression and serves to prime them for future reactivation, a phenomenon called transcriptional memory. We have found that a mechanistically similar phenomenon occurs in human cells. A subset of interferon γ-inducible genes are much more strongly induced in cells that have previously been exposed to IFNγ. As in yeast, this type of transcriptional memory involves a physical interaction with Nups, binding of a poised form of RNA polymerase II to the promoter and changes in chromatin structure. However, unlike yeast, the interaction of these genes with Nups occurs in the nucleoplasm. Finally, in cells transiently knocked down for Nup98, memory is lost; RNA polymerase II does not remain associated with IFNγ-inducible promoters and the rate of reactivation is dramatically reduced. These results suggest that Nup98 (Nup100 in yeast) plays a conserved role in promoting epigenetic transcriptional memory.

172

In vivo three-dimensional characterization of mRNA export through the nuclear pore complex.

W. Yang1; 1Temple University, Philadephia, PA

Protein-receptor-facilitated nuclear export of messenger RNAs (mRNAs) through the nuclear pore complexes (NPCs) is a key step for the flow of genetic information in eukaryotic cells. However, the transport kinetics, the three-dimensional (3D) pathway and the export selectivity mechanism of mRNA-protein complexes through the NPCs remain poorly understood, although they are of fundamental interest. Here we employed a super-resolution microscopy imaging approach to three-dimensionally characterize mRNAs exporting through individual NPCs in living human cells. With a spatiotemporal resolution of 2 ms and 8 nm, the fundamental features of mRNAs export through the NPCs, escaped from previous measurements of electron microscopy and single-molecule fluorescence microscopy, have been obtained: 1) mRNA complexes were decelerated and then selected at the narrowest waist of the NPC as they entered the NPC from the nucleus. Approximately one third of these incoming complexes successfully arrived at the cytoplasm, and the others aborted their export and returned back to the nucleus; 2) either the successful or the abortive mRNA export events took approximately 12 ms to interact with the NPCs; 3) In a 3D view, mRNA complexes primarily paved their passageways through the periphery around a rarely occupied central axial conduit on the nucleoplasmic side and in the central channel of the NPC, then dissociated on the cytoplasmic side of the NPC, and finally passively diffused into the cytoplasm.

173

In vivo visualization of chromosome synapsis in C. elegans.

O. Rog1, A. F. Dernburg1; 1University of California, Berkeley; and Howard Hughes Medical Institute, Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences (QB3), Berkeley, CA

Meiosis is the special cell division process that enables the production of haploid gametes. During meiotic prophase, chromosomes form linkages with their homologous partners to enable reductional segregation during the first meiotic division. Essential to this process is the pairwise alignment of homologous chromosomes along their entire lengths. In most eukaryotes this alignment is reinforced through synapsis, the polymerization of a structurally conserved structure called the synaptonemal complex (SC), which links homologous chromosomes. The SC promotes genetic exchanges (crossovers) between homologs, and likely regulates their number and location. Synapsis is a dynamic process, the details of which are difficult to infer from images of fixed cells or tissues. Our goal has been to better understand this process through analysis in living nematodes, using fluorescently tagged SC components and high- resolution time-lapse microscopy. Our observations have revealed that synapsis is rapid and highly processive. Individual chromosomes achieve full synapsis in 20-30 minutes, at a rate of ~170nm per minute. We find that initiation of synapsis is a relatively infrequent event that is rate-limiting for completion of synapsis, consistent with evidence that initiation is subject to strict regulation. Initiation occurs asynchronously on each chromosome, resulting in staggered synapsis of different chromosomes. In C. elegans, synapsis is accompanied by rapid chromosome motions driven by dynein, which is coupled through the nuclear envelope to one end of each chromosome. Under conditions where this motion is severely abrogated, synapsis remains processive but is ~5-fold slower. This suggests that chromosome motion promotes

homolog alignment, enabling synapsis to proceed more rapidly. Moreover, it reveals a novel function for the rapid chromosome motions that have been observed in diverse organisms during meiotic prophase.

174

Assembly, dynamics and function of the synaptonemal complex (SC) in meiotic prophase nuclei.

W. Zhang1, D. Libuda1, S. Mlynarczyk-Evans1, D. Pattabiraman1, G. Chen1, M. Presler1, A. M. Villeneuve1; 1Stanford University, Stanford, CA

The SC is a highly ordered proteinaceous structure that assembles at the interface between paired and aligned homologous chromosomes during meiotic prophase. The SC has long been recognized as a hallmark feature of the meiotic program and has at least two conserved functions, in stabilizing tight associations along the lengths of homologs and in promoting maturation of recombination intermediates into crossovers. Recent work has shown that the SC also plays a role in inhibiting crossover formation during C. elegans meiosis, thereby limiting crossover number. Further, SC structures undergo patterned disassembly that creates an organization crucial for directing the pattern of chromosome segregation during the meiotic divisions. In addition, several lines of evidence suggest that SC assembly is part of a feedback mechanism that regulates prophase progression to assure a successful meiotic outcome.

While substantial progress has been made in identifying SC components and defining its biological roles, the SC has remained enigmatic in many ways. Although the SC normally assembles only between aligned homologous chromosomes, the SC structure itself is indifferent to homology. Moreover, SC assembly appears to be cooperative and processive, indicating a requirement for tight coordination of homolog pairing and synapsis. Further, the zipper-like appearance of the SC in EM images belies a growing realization that the SC structure is likely much more dynamic than previously appreciated. We will discuss mechanisms that regulate assembly of the SC to ensure that it only occurs in a productive manner, i.e., linking the axes of correctly paired and aligned homologous chromosomes. We will show that reducing the pool of specific SC subunits results in attenuated crossover interference, reflected in an increase in the number of cytologically-differentiated crossover sites, implying a requirement for appropriate stoichiometry to confer normal SC function. Further, we will present evidence from FRAP analysis that the ″fully assembled″ SC structure is indeed dynamic, undermining the view of the SC as a static scaffold.

175

Corralling the microtubule tip: Nanoscale kinetochore architecture suggests an integrative model for its bidirectional motility.

P. Aravamudhan1, A. P. Joglekar2; 1Biophysics, University of Michigan, Ann Arbor, MI, 2Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI

The kinetochore is a macromolecular machine that drives chromosome movement and segregation during cell division. The molecular mechanism underlying this unique, bidirectional motor depends as much on its nanoscale protein architecture as the structures and biochemical properties of individual proteins. Yet, the in vivo kinetochore architecture, and more specifically, the nanoscale distribution of individual microtubule (MT) binding protein molecules around and along the microtubule tip remains unknown. To define this architecture, we have developed a systematic FRET quantification technique in live cells. Our analysis reveals two microtubule- binding domains within the budding yeast (Saccharomyces cerevisiae) kinetochore. The first unit in this bipartite arrangement consists of Ndc80 and Dam1. The MT-binding domains of

these complexes work in close spatial proximity (< 10 nm), suggesting a functional integration of these molecules. We propose that this force generating unit is mainly responsible for MT depolymerization coupled movement. The MT-associated, plus-end tracking protein Stu2p (XMAP215) makes up the second unit. The C-terminus of this large protein situated ~ 35 nm behind Ndc80-Dam1. This multi-function protein likely participates in polymerization-coupled motility. We propose that it contributes either tip-tracking ability or destabilizes the growing tip. Thus, our reconstructed kinetochore architecture, when considered in the context of the unique structural and MT-binding properties of each individual protein, suggests an integrative model explaining bidirectional kinetochore movement that is tightly coupled with MT polymerization and depolymerization. This scheme of corralling the dynamic MT tip is likely conserved in all eukaryotes, even if organisms freely substitute proteins with analogous activities (with the exception of the Ndc80 complex) to fine-tune chromosome motility.

176

The structure and function of a chromatin spring in mitosis.

K. S. Bloom1, A. Stephens1, J. Verdaasdonk1, J. Haase1, E. Yeh1; 1Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC

The physical properties of a long-chain polymer such as DNA and its organization inside the cell contribute in fundamental ways to replication, repair, transcription and chromosome segregation. DNA and the unique chromatin organization at the centromere function as a mechanical spring in mitosis. We apply principles of polymer physics to understand how chromatin architecture (such as DNA wrapping around nucleosomes, looping and catenation) contributes to faithful chromosome segregation. Cohesin and condensin together with pericentric chromatin constitute the spring that functions as a restoring force counterbalancing the microtubule extensional force in metaphase of mitosis. The spring exhibits asymmetric stretching, inconsistent with a simple linear spring model. We present experimental and modeling evidence for a threshold extension in the spring to switch between cohesin-condensin rich versus depleted spring constants, that accurately recapitulates in vivo chromatin and spindle dynamics.