Jagesh V. Shah

Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury

Fibrosis is responsible for chronic progressive kidney failure, which is present in a large number of adults in the developed world. It is increasingly appreciated that acute kidney injury (AKI), resulting in aberrant incomplete repair, is a major contributor to chronic fibrotic kidney disease. The mechanism that triggers the fibrogenic response after injury is not well understood. In ischemic, toxic and obstructive models of AKI, we demonstrate a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome. G2/M-arrested proximal tubular cells activate c-jun NH2-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production. Treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney. Hence, epithelial cell cycle arrest at G2/M and its subsequent downstream signaling are hitherto unrecognized therapeutic targets for the prevention of fibrosis and interruption of the accelerated progression of kidney disease.

Waiting for Anaphase: Mad2 and the Spindle Assembly Checkpoint

Although all the pieces of the puzzle may seem to be in place, there are still several key processes that have been widely posited but remain experimentally unverified. What attracts Mad2 to the kinetochore and what causes its release? Is it Mad1, as has been hypothesized from its apparent loss (along with Mad2) from the kinetochore after microtubule attachment (Figure 3AFigure 3A)? A pair of reports reveal a fly in the ointment (3xBasto, R., Gomes, R., and Karess, R.E. Nat. Cell Biol. 2000; 2: 939–943Crossref | PubMed | Scopus (91)See all References, 5xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all References). Specifically, they demonstrate that the zeste-white 10 protein (Zw10) and its binding partner, rough deal (Rod), both originally discovered in Drosophila and without homologs in yeast, are required for activation of the spindle checkpoint in human cells and flies. These proteins have been previously shown to recruit dynein and dynactin to the kinetochore and, upon microtubule attachment, to “stream” along kinetochore microtubules to the spindle poles—an attractive mechanism for turning the checkpoint off. So it comes as a big surprise that disruption of Zw10 or Rod function genetically (Basto et al. 2000xBasto, R., Gomes, R., and Karess, R.E. Nat. Cell Biol. 2000; 2: 939–943Crossref | PubMed | Scopus (91)See all ReferencesBasto et al. 2000) or with antibodies (Chan et al. 2000xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all ReferencesChan et al. 2000) did not arrest cells in mitosis but instead prevented cells from activating the checkpoint in the presence of spindle damage. In human cells, antibody inhibition of Zw10 or Rod function leaves Mad2, the trusty wait signal, still present at the kinetochores of the unattached chromosomes even though the checkpoint is off (Chan et al. 2000xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all ReferencesChan et al. 2000)!Figure 3Unresolved Questions in the Spindle CheckpointFeatures of checkpoint signaling yet to be experimentally verified include (A) what is Mad2*, (B) what deactivates it, and (C) how do the components upstream of Mad2 generate Mad2* at unattached kinetochores and how is such signaling silenced upon microtubule attachment?View Large Image | View Hi-Res Image | Download PowerPoint SlideHow could this be? One possible interpretation is that some proteins may be required to bring Mad2 to the kinetochore (e.g., Mad1), whereas others, Zw10 or Rod for example, may be responsible for releasing Mad2*. If this is the case, Zw10 and Rod must only be important for releasing Mad2* from unattached kinetochores because Mad2 is released from attached kinetochores despite the loss of Zw10 and Rod (Figure 3AFigure 3A). This hypothesis is now directly testable—does Mad2 fail to cycle at unattached kinetochores in Zw10 or Rod disrupted cells?Determining the components of the inhibitory signal (Figure 3BFigure 3B) will firmly establish the molecular link between the unattached kinetochore and the inhibition of anaphase. The bigger question, of course, is how these elements are coordinated upstream of Mad2 and downstream of spindle attachment (Figure 3CFigure 3C). Attachment of microtubules to the kinetochore is likely to be mediated by dynein and kinesin motors (e.g., CENP-E, XKCM1/MCAK; for review see Sharp, et al. 2000xSharp, D.J., Rogers, G.C., and Scholey, J.M. Nature. 2000; 407: 41–47Crossref | PubMed | Scopus (364)See all ReferencesSharp, et al. 2000), especially CENP-E, a kinetochore kinesin that can directly bridge between spindle microtubules and the checkpoint kinase BubR1 (4xChan, G.K., Jablonski, S.A., Sudakin, V., Hittle, J.C., and Yen, T.J. J. Cell Biol. 1999; 146: 941–954Crossref | PubMed | Scopus (269)See all References, 19xYao, X., Abrieu, A., Zheng, Y., Sullivan, K.F., and Cleveland, D.W. Nat. Cell Biol. 2000; 2: 484–491Crossref | PubMed | Scopus (254)See all References) and which can be essential for kinetochore signaling in vitro (Abrieu et al. 2000xAbrieu, A., Kahana, J.A., Wood, K.W., and Cleveland, D.W. Cell. 2000; 102: 817–826Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesAbrieu et al. 2000). Microtubule attachment leads to even more cascades that will undoubtedly involve mitotic kinases such as Mps1, MAP kinase, Bub1, and BubR1 and the little talked about phosphatases such as budding yeast Glc7/PP1. In addition, proteins such as Bub3, Mad3, Mad1, and Zw10/Rod all play an as yet undefined role in the generation, release, regulation, and/or composition of the wait signal. Stay tuned. Much more remains to be unraveled.‡To whom correspondence should be addressed (e-mail: dcleveland@ucsd.edu).

THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea–induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat–containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88–enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.

Identification of Signaling Pathways Regulating Primary Cilium Length and Flow-Mediated Adaptation

The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, it is expected the development of a potent, selective small molecule inhibitor of Mps1 would greatly facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 co-crystal structures of novel, selective small molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit, and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells possessing extra centrosomes, an abnormality found in some cancers, Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Defects in the IFT-B Component IFT172 Cause Jeune and Mainzer-Saldino Syndromes in Humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

The ciliary transition zone: from morphology and molecules to medicine.

Researchers from various disciplines, including cell and developmental biology, genetics and molecular medicine, have revealed an exceptional diversity of cellular functions that are mediated by cilia-dependent mechanisms. Recent studies have directed our attention to proteins that localize to the ciliary transition zone (TZ), a small evolutionarily conserved subcompartment that is situated between the basal body (BB) and the more distal ciliary axoneme. These reports shed light on the roles of TZ proteins in ciliogenesis, ciliary protein homeostasis and specification of ciliary signaling, and pave the way for understanding their contribution to human ciliopathies. In this review, we describe the interplay of multimeric protein complexes at the TZ, integrating morphological, genetic and proteomic data towards an account of TZ function in ciliary physiology.

Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

Centromere protein F (CENP‐F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore‐bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP‐F does not affect cell cycle progression in S and G2. In a subset of CENP‐F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis‐segregation. In contrast, the majority of CENP‐F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi‐oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP‐marked Mad1 reveals that throughout the mitotic delay some aligned, CENP‐F depleted kinetochores continuously recruit Mad1. Others rebind YFP‐Mad1 intermittently so as to produce ‘twinkling’, demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP‐F in efficient assembly of a stable microtubule–kinetochore interface.

Slow axonal transport: fast motors in the slow lane

The bulk of neuronally synthesized proteins destined for the axon is transported in a phase of transport approximately 100 times slower (1mm/day) than the vesicular traffic of fast axonal transport (100mm/day). Of late, a number of studies have shed considerable light on the controversies and mechanisms surrounding this slow phase of axonal transport. Along-standing controversy has centered on the form of the transported proteins. One major transport cargo, neurofilament protein, has now been seen in a number of contexts to be transported primarily in a polymeric form, whereas a second cargo tubulin is transported as a small oligomer. The development of techniques to visualize the slow transport process in live cells has demonstrated that instantaneous motions of transported neurofilaments, and presumably other slow transport cargoes, are fast, bidirectional and interspersed with long pauses. This and additional biochemical efforts indicate that traditional fast motors, such as conventional kinesin and dynein, are responsible for these fast motions.

Strain hardening of fibrin gels and plasma clots

Biological macromolecules have unique rheological properties that distinguish them from common synthetic polymers. Among these, fibrin has been studied extensively to understand the basic mechanisms of viscoelasticity as well as molecular mechanisms of coagulation disorders. One aspect of fibrin gel rheology that is not observed in most polymeric systems is strain hardening: an increase in shear modulus at strain amplitudes above 10%. Fibrin clots and plasma clots devoid of platelets exhibit shear moduli at strains of approximately 50% that are as much as 20 times the moduli at small strains. The strain hardening of fibrin gels was eliminated by the addition of platelets, which caused a large increase in shear storage modulus in the low strain linear viscoelastic limit. The reduction in strain hardening may result from fibrin strand retraction which occurs when platelets become activated. This interpretation is in agreement with recent theoretical treatments of semi-flexible polymer network viscoelasticity.