Yixian Zheng, Ph.D. Staff Member Department of Embryology, Carnegie Institution of Washington Adjunct Professor Department of Biology CMDB Graduate Program Faculty B.S. (Biology) Sichuan University, PR China Ph.D. (Molecular Genetics) Ohio State University

[Research Interests] [Representative Publications]

RESEARCH INTERESTS

Cell Division, Cell Morphogenensis, and Cell Fate Specification

My lab is interested in understanding how the cytoskeleton, the nuclear lamina, and the membrane network coordinate with one another to regulate cell division and differentiate.  We employ a variety of model systems to study the mechanism of cell division.  Using embryonic stem cells (ESC) and mouse embryos, we also study how cell migration, cellular morphogenesis, and cell division are coupled to cell fate specifications during development. 

Cell division—mitotic spindle assembly and chromosome segregation
Spindle morphogenesis ensures equal segregation of mitotic chromosomes and proper partitioning of other cellular components important for the survival, proliferation, and differentiation of daughter cells. Spindle assembly and organization is orchestrated by a large array of structural and regulatory proteins. 

Centrosome and MT nucleation
Centrosomes play important roles in mitotic spindle assembly by providing the major MT nucleation and organization sites. Through the purification and study of the ~36S γ-tubulin ring complex (γTuRC), we showed that this complex is essential not only for MT nucleation at the centrosome but also for mitotic spindle assembly. 

Using a centrosome-complementation assay, biochemical fractionations, and mass spectrometry (in collaboration with Dr. John Yates), we have identified additional candidate factors that regulate MT nucleation from centrosomes.  By further characterizing one of these proteins called Pontin, an AAA+ ATPase involved in diverse array of cellular functions, we have shown that Pontin interacts with γTuRC to promote MT assembly in mitosis.

Spindle assembly—the RanGTPase signaling and the mitotic spindle matrix
We have shown that the nuclear small GTPase Ran regulates multiple aspects of spindle assembly in mitosis by modulating the interaction between spindle assembly factors (SAFs) containing nuclear localization signals (NLS) with nuclear transport receptors importin α and β.  Additional analyses have led us to uncover an important Ran signaling pathway that leads to activation of the important mitotic kinase Aurora A (AurA) (Fig. 2). By coupling AurA to magnetic beads (AurA-beads), we developed an efficient spindle assembly assay, which has offered us with a great opportunity to study not only AurA kinase but also spindle assembly (Fig. 3).

Aided by this assay, we uncovered a mitosis-specific function for lamin B, a type V intermediate filament protein with a well-established role in nuclear organization and gene regulation in interphase.  We have shown that lamin B is a downstream target of RanGTPase and it regulates spindle morphogenesis as one of the structural components of the membranous spindle matrix that tethers a number of SAFs. Our studies suggest that RanGTP independently regulates the assembly of MTs and lamin B, which reciprocally regulate each other through interacting with the SAFs, leading to spindle assembly.

Spindle Assembly—the RanGTPase signaling and the cell cycle
Our studies have shown that the cell cycle machinery directly regulates the Ran-signaling pathway by phosphorylating the NLS of RCC1, the nucleotide exchange factor for Ran.  Phosphorylated RCC1 does not bind to importins α and β, consequently is able to produce a high concentration of RanGTP on chromosomes to guide spindle assembly toward the chromosomes. By manipulating a Ran binding protein called RanBP1 in combination with computational simulation (collaboration with Dr. Pablo Iglesias), we showed that the highest RanGTP concentration gradient could be achieved when all mitotic chromosomes have congressed to the metaphase plate.  This elevated RanGTP level facilitates metaphase to anaphase transition by aiding the inactivation of the spindle checkpoint.  Together, our studies have demonstrated that the Ran signaling pathway and the cell cycle machinery reciprocally regulate each other to control both spindle assembly and mitotic progression.

Chromosome segregation and spindle disassembly
We found that two sets of membrane-associated enzymes, the ubiquitin selective chaperone called the p97-Ufd1-Npl4 complex and the deubiquitination enzyme called FAM, regulate mitosis. The p97-Ufd1-Npl4 complex and FAM interact with and regulate Survivin, a subunit of the chromosome passenger complex required for chromosome segregation and cytokinesis.  Whereas FAM removes the K63-ubiquitin linkage on Survivin in mitosis, p97-Ufd1-Npl4 is required for Survivin to acquire such linkages.  A balanced K-63 ubiquitination level on Survivin in turn regulates the dynamic binding of Survivin to centromeres, which is important for chromosome alignment and segregation. Our further study of p97-Ufd1-Npl4 has demonstrated that this chaperone also regulates spindle disassembly at the end of mitosis. 

Lineage specification during development and stem cell differentiation   
Our studies of mitosis have shown that the component of the nuclear lamina (lamin B) functions with the MT cytoskeleton to help chromosome organization and movement on the mitotic spindle. By analogy, the interphase nuclear lamina might also function with cytoskeletons to organization chromatin and to affect gene expression. Indeed, the interphase nuclear envelope and lamina make extensive contacts with chromatin and cytoskeleton in the nucleus and cytoplasm, respectively. Therefore, cytoskeleton re-organization occurring during cell migration and cell shape change could influence chromatin organization through the nuclear lamina, which in turn could affect gene regulation. The changes in transcription may also affect cytoskeleton assembly dynamics. This kind of reciprocal regulation could play an important role in development because specification of cell lineages relies not only on transcriptional regulation but also on cell division and gradual cellular morphological changes.

We have explored this idea by performing live imaging of the behavior of pluripotent ESCs as they differentiate into different cell lineages. We found that distinct cell behavior accompanied different differentiation pathways. We have focused our study on the first lineage specification in pre-implantation mammals, which is known to involve cell sorting and transcriptional changes. Successful specification of the first lineage results in the formation of a blastocyst containing the outer trophectoderm (TE) cells expressing the transcription factor Cdx2 and the inner cell mass (ICM) that could give rise to ESCs. 

By analyzing cellular morphogenesis and gene expression profile during TE differentiation from mouse ESCs, we have uncovered a new morphological regulator that indeed could influence the expression of Cdx2.  We believe further study of the function of this new regulator should shed light on how cell morphogenesis is coupled with transcriptional changes during differentiation.  

REPRESENTATIVE PUBLICATIONS

1.    Zheng Y, Jung MK, & Oakley BR (1991). γ-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome.  Cell 65, 817-823.

2.    Zheng Y, Wong ML, Alberts B, & Mitchison TJ (1995). A γ-tubulin ring complex from the unfertilized egg of Xenopus laevis can nucleate microtubule assembly in vitro. Nature 378, 578-583. (Research Article; News & Views: Oakley, Nature 378, 555-556)

3.    Wilson PG, Zheng Y, Oakley CE, Oakley BR, Borisy GG, & Fuller MT (1997). Differential expression of two γ-tubulin isoforms during gametogenesis and development in Drosophila.  Developmental Biology 184, 207-221.

4.    Dictenberg JB, Zimmerman W, Sparks CA, Young A, Vidair C, Zheng Y, Carrington W, Fay FS, & Doxsey SJ (1998). Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into A Novel Lattice at the Centrosome.  Journal Cell Biology 141, 163-174.

5.    Martin O, Gunawardane R., Iwamatsu A, & Zheng Y (1998). Xgrip109: A γ-tubulin associated protein with an essential role in γTuRC assembly and centrosome function. Journal of Cell Biology 141, 675-687.

6.    Moritz M, Zheng Y, Alberts B, & Oegema K (1998). Recruitment of the γ-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. Journal of Cell Biology 142, 775-786.

7.    Zheng Y, Wong ML, Alberts B, & Mitchison T (1998). Purification and assay of γ-tubulin ring complex.  Methods in Enzymology 298, Part B, 218-228.

8.    Field CM, Oegema K., Zheng Y, Mitchison T, & Walczak CE (1998). Purification of Cytoskeletal Proteins Using Peptide Antibodies. Methods in Enzymology 298, Part B, 525-541.

9.    Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison T, & Zheng Y (1999). Characterization of Two Related Drosophila γ-tubulin Complexes that Differ in Their Ability to Nucleate Microtubules.  Journal of Cell Biology 144, 721-733.

10.    Wiese C & Zheng Y (1999). γ-Tubulin Complexes and Their Interaction with Microtubule Organizing Centers.  Current Opinion in Structural Biology 9, 250-259.

11.    Wilde A & Zheng Y (1999). Stimulation of Microtubule Aster Formation and Spindle Assembly in Xenopus Egg Extracts by the Small GTPase Ran. Science 284, 1359-1362. (News Focus: Pennisi, Science 284, 1260-1261, 1999; Commentary: Desai & Hyman, Current Biology 9, R704-707, 1999)

12.    Wiese C & Zheng Y (2000). A New Function for the γ-tubulin Ring Complex as a Microtubule Minus-end Cap.  Nature Cell Biology 2, 358-364. (News & Views: Erickson, Nature Cell Biology 2, E93-E96)

13.    Zhang L, Keating T, Wilde, A, Borisy G, & Zheng Y (2000). The Role of Xgrip210 in γ-Tubulin Ring Complex Assembly and Centrosome Recruitment. Journal of Cell Biology 151, 1525–1535.

14.    Gunawardane R, Martin O, Cao K, Zhang L, Dej K, Iwamatsu A, & Zheng Y (2000). Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits.  Journal of Cell Biology 151, 1513–1523.

15.    Gunawardane R, Lizarraga S, Wiese C, Wilde A, & Zheng Y (2000). γ-Tubulin Complexes and Their Role in Microtubule Nucleation.  In The Centrosome in Cell Replication and Early Development. pp 55-73. Academic Press (Book).

16.    Gunawardane RN, Lizarraga SB, Wiese C, Wilde A, & Zheng Y (2000). γ-Tubulin Complexes and Their Role in Microtubule Nucleation.  Current Topics in Developmental Biology 49, 55-73.

17.    Wilde A, Lizarraga S, Zhang L, Wiese C, Gliksman N, Walczak C, & Zheng Y (2001). Ran stimulates spindle assembly by changing microtubule dynamics and the balance of motor activities.  Nature Cell Biology 3, 221-227. (News & Views: Walczak, Nature Cell Biology 3, E69-70, 2001)

18.    Wiese C, Wilde A, Adam S, Moore M, Merdes A, & Zheng Y (2001). Role of Importin-β in Coupling Ran to Downstream Targets in Microtubule Assembly.  Science 291, 653-656. (News & Views: Walczak, Nature Cell Biology 3, E69-70, 2001)

19.    Gunawardane RN, Zheng Y, Oegema K, & Wiese C (2001). Purification and reconstitution of Drosophila gamma-tubulin complexes.  Methods in Cell Biology 67, 1-25.

20.    Lizarraga SB, Zheng Y, & Wilde AR (2002). Characterization of the effects of RanGTP on the microtubule cytoskeleton. Methods in Molecular Biology 189, 247-260.

21.    Gunawardane R, Martin OC, & Zheng Y (2003). Characterization of a new γTuRC subunit with WD repeats.  Molecular Biology of the Cell 14, 1017-1026.

22.    Tsai MY, Wiese C, Cao K, Martin OC, Donovan P, Ruderman J, Prigent C, & Zheng Y (2003). A Ran-signaling pathway mediated by the mitotic kinase Aurora A in spindle assembly.  Nature Cell Biology 5, 242-248.

23.    Li HY, Wirtz D, & Zheng Y (2003). A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo. Journal of Cell Biology 160, 635-644.

24.    Li HY, Cao K, & Zheng Y. (2003) Ran in spindle checkpoint: a new function for a versatile GTPase. Trends in Cell Biology 13, 553-557.

25.    Cao K, Nakajima R, Meyer HH, & Zheng Y. (2003). The AAA-ATPase Cdc48/p97 regulates spindle disassembly at the end of mitosis. Cell 115, 355-367. (Highlight: Nature Reviews Molecular Cell Biology 4, 906, 2003; Commentary: Cheeseman and Desai, Current Biology 14, R70-72, 2004)

26.    Ems-McClung SC, Zheng Y, & Walczak CE (2004). Importin / and Ran-GTP Regulate XCTK2 Microtubule Binding through a Bipartite Nuclear Localization Signal.  Molecular Biology of the Cell 15, 46-57.

27.    Kawaguchi S & Zheng Y (2004). Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP.  Molecular Biology of the Cell 15, 37-45.

28.    Li HY & Zheng Y (2004). Mitotic phosphorylation of RCC1 is essential for RanGTP gradient production and spindle assembly in mammalian cells. Genes and Development 18, 512-527.

29.    Cao K & Zheng Y (2004). The Cdc48/p97-Ufd1-Npl4 Complex: Its Potential Role in Coordinating Cellular Morphogenesis during the M-G1 Transition. Cell Cycle 3, 422-424.

30.    Li HY & Zheng Y (2004). The Production and Localization of GTP-Bound Ran in Mitotic Mammalian Tissue Culture Cells. Cell Cycle 3, 993-995.

31.    Ducat DC & Zheng Y (2004). Aurora kinases in spindle assembly and chromosome segregation. Experimental Cell Research 301, 60-67.

32.    Nakajima, R, Tsai, M-Y, & Zheng Y (2004). Centrosomes and Microtubule Nucleation, Encyclopedia of Biological Chemistry 1, 372-376. W. J. Lennarz and M. D. Lane (Ed), Elsevier Inc.

33.    Zheng Y (2004). G Protein Control of Microtubule Assembly, Annual Review of Cell and Developmental Biology 20, 867-894.

34.    Tsai M-Y & Zheng Y (2005). Aurora A Kinase-Coated Beads Function as Microtubule-Organizing Centers and Enhance RanGTP-Induced Spindle Assembly.  Current Biology 15, 2156-2163.

35.    Vong QP, Cao K, Li HY, Iglesias PA, & Zheng Y (2005). Chromosome Alignment and Segregation Regulated by Ubiquitination of Survivin. Science 310, 1499-1504. (Perspective: Earnshaw, Science 310, 1443-1444)

36.    Tsai M-Y, Wang S, Heidinger JM, Shumaker D, Adam SA, Goldman RD, & Zheng Y (2006). A Mitotic Lamin B Matrix Induced by RanGTP Required for Spindle Assembly.  Science 311, 1887-1893. (Research Article; News & Views: Hayes, Nature Cell Biology 8, p550, 2006; Research Highlight: Nature Reviews Molecular Cell Biology 7, p307, 2006).

37.    Goodman B & Zheng Y (2006). Mitotic spindle morphogenesis: Ran on the microtubule cytoskeleton and beyond.  Biochemical Society Transactions 34, 716-721.

38.    Wiese C & Zheng Y (2006). Microtubule nucleation: γ-tubulin and beyond.  Journal of Cell Science 119, 4143-4153.

39.    Zheng Y & Tsai M-Y (2006). The Mitotic Spindle Matrix: A Fibro-Membranous Lamin Connection.  Cell Cycle 5, 2345-2347.

40.    Spradling AC & Zheng Y (2007). The Mother of All Stem Cells? Science 315, 469-470.

41.    Liu Z, Vong QP, & Zheng Y (2007). CLASPing Microtubules at the trans-Golgi Network. Developmental Cell 12, 839-840.

42.    Li HY, Ng WP, Wong CH, Iglesias PA, & Zheng Y (2007). Coordination of Chromosome Alignment and Mitotic Progression by the Chromosome-Based Ran Signal.  Cell Cycle 6, 1886-1895.

43.    Channels WE, Nedelec FJ, Zheng Y, & Iglesias PA (2008). Spatial regulation improves anti-parallel microtubule overlap during mitotic spindle assembly. Biophys Journal 94, 2598-609.

44.    Zheng Y & Oegema K (2008). Cell Structure and Dynamics. Current Opinion in Cell Biology 20, 1–3.

45.    Ducat D, Kawaguchi S, Liu H, Yates JR 3rd, & Zheng Y (2008). Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin. Molecular Biology of the Cell, 19:3097-3110.

46.    Wilde A & Zheng Y (2008). Ran out of the nucleus for apoptosis. Nature Cell Biology 11, 11-12.

47.    Li M, Tsai MY, Lu B, Chen R, Yates III JR, Zhu X, & Zheng Y (2009).  A Requirement of Nudel and Dynein for Spindle Matrix Assembly during Spindle Morphogenesis.  Nature Cell Biology 11, 247-256

48.    Martin O, DeSevo CG, Guo BZ, Koshland DE, Dunham MJ, & Zheng Y (2009). Telomere behavior in a hybrid yeast. Cell Research 19, 910-912

49.    Liu Z & Zheng Y (2009).  A Requirement for Epsin in Mitotic Membrane and Spindle Organization.  Journal of Cell Biology 186, 473-480

50.    Bembenek JN, White JG, and Zheng Y (2009).  A role for separase in the regulation of Rab-11-positive vesicles at the cleavage furrow and midbody. Current Biology (in revision).

51.    Goodman B, Channels V, Iglesias, P, and Zheng Y (2009).  Lamin B restrains the activity of kinesin Eg5 in spindles assembled in Xenopus egg extracts. Journal of Cell Biology (under review).

52.    Vong QV, Liu Z, Yoo JG, Chen R, Xie W, Sharov AA, Fan C-M, Ko MSH, Zheng Y (2009).  Borg5 regulates Cdx2 expression and cell morphogenesis during trophectoderm differentiation.  Submitted.

Lab Members

Postdoctoral Fellows:
Chris Wiese
Andy Wilde
 
Graduate Students:
Kan Cao

Daniel Ducat 
 
Research Technicians:
Ona Martin
Lijun Zhang