Papers by David Michaelson
Worm, 2014
The DNA content of nuclei is a valuable measure of cell cycle status. Irises is a software tool t... more The DNA content of nuclei is a valuable measure of cell cycle status. Irises is a software tool to facilitate systematic in situ determination of DNA content for cell cycle analysis at single-nucleus resolution within complex tissues. We demonstrate the utility of the tool with analysis of DNA content in germline nuclei of C. elegans. Compared with results obtained by manual analysis, we find the tool greatly facilitates analysis by improving speed at least 5-fold while maintaining accuracy. The source code and instruction manual (including installation for both Mac and PC) are provided.
Rac1L61/Ktail cannot enter the nucleus. Rac1L61ΔAAX is entirely nuclear. (A) Cytofluorimetry indi... more Rac1L61/Ktail cannot enter the nucleus. Rac1L61ΔAAX is entirely nuclear. (A) Cytofluorimetry indicates equivalent expression levels. (B) TRITC-phalloidin staining reveals that although Rac161L/Ktail induces marked peripheral ruffling like Rac1L61, Rac1L61ΔAAX does not. Bar, 10 μm. (C) Mitotic index (mitoses per number of cells over 16 h) of cells transduced with the indicated constructs (mean ± SEM; = 3).<b>Copyright information:</b>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"The Journal of Cell Biology 2008;181(3):485-496.Published online 5 May 2008PMCID:PMC2364699.
Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remai... more Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCHmutant, to delineate NOTCH-driven tumor responses to radiotherapy. At20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor pheno-type in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.
Developmental Biology, 2016
Many organisms accumulate a pool of germline stem cells during development that is maintained in ... more Many organisms accumulate a pool of germline stem cells during development that is maintained in later life. The dynamics of establishment, expansion and homeostatic maintenance of this pool are subject to both developmental and physiological influences including the availability of a suitable niche microenvironment, nutritional status, and age. Here, we investigated the dynamics of germline proliferation during stages of expansion and homeostasis, using the C. elegans germ line as a model. The vast majority of germ cells in the proliferative zone are in interphase stages of mitosis (G1, S, G2) rather than in the active mitotic (M) phase. We examined mitotic index and DNA content, comparing different life stages, mutants, and physiological conditions. We found that germ cells in larval stages cycle faster than in adult stages, but that this difference could not be attributed to sexual fate of the germ cells. We also found that larval germ cells exhibit a lower average DNA content compared to adult germ cells. We extended our analysis to consider the effects of distance from the niche and further found that the spatial pattern of DNA content differs between larval and adult stages in the wild type and among mutants in pathways that interfere with cell cycle progression, cell fate, or both. Finally, we characterized expansion of the proliferative pool of germ cells during adulthood, using a regeneration paradigm (ARD recovery) in which animals are starved and re-fed. We compared adult stage regeneration and larval stage expansion, and found that the adult germ line is capable of rapid accumulation but does not sustain a larval-level mitotic index nor does it recapitulate the larval pattern of DNA content. The regenerated germ line does not reach the number of proliferative zone nuclei seen in the continuously fed adult. Taken together, our results suggest that cell cycle dynamics are under multiple influences including distance from the niche, age and/or maturation of the germ line, nutrition and, possibly, latitude for physical expansion.
Methods in Enzymology, 2006
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the stu... more The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems.
Methods in Enzymology, 2002
Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the... more Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mam- malian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic gran- ules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specilic rabbit defensin cDNAs that code for
Journal of Cell Biology, 2008
Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus ... more Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane–targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle–blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cy...
Journal of Cell Biology, 2001
Determinants of membrane targeting of Rho proteins were investigated in live cells with green flu... more Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected b...
Oncogene, 2005
Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and me... more Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells.
Molecular Biology of the Cell, 2002
Membrane targeting of G-protein αβγ heterotrimers was investigated in live cells by use of Gα and... more Membrane targeting of G-protein αβγ heterotrimers was investigated in live cells by use of Gα and Gγ subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gβγ contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Gγ1, but not geranylgeranylated Gγ2, required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Gγ with the fatty acyl modifications of Gα. Gα associated with Gβγ on the Golgi and palmitoylation of Gα was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane.
Molecular Biology of the Cell, 2005
The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting bec... more The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on...
FEBS Letters, 1993
Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils... more Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and-2) or Paneth cells (HD-5) are organized differently: HD-5 has only two exons, and MCP-1 and-2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression.
Development, 2010
Cell proliferation must be coordinated with cell fate specification during development, yet inter... more Cell proliferation must be coordinated with cell fate specification during development, yet interactions among pathways that control these two critical aspects of development are not well understood. The coordination of cell fate specification and proliferation is particularly crucial during early germline development, when it impacts the establishment of stem/progenitor cell populations and ultimately the production of gametes. In C. elegans, insulin/IGF-like receptor (IIR) signaling has been implicated in fertility, but the basis for the fertility defect had not been previously characterized. We found that IIR signaling is required for robust larval germline proliferation, separate from its well-characterized role in preventing dauer entry. IIR signaling stimulates the larval germline cell cycle. This activity is distinct from Notch signaling, occurs in a predominantly germline-autonomous manner, and responds to somatic activity of ins-3 and ins-33, genes that encode putative insu...
Cell, 1999
the CAAX motif substitutes for palmitoylated cysteines in acting as a second required PM targetin... more the CAAX motif substitutes for palmitoylated cysteines in acting as a second required PM targeting signal (Han-Departments of Medicine and Cell Biology New York University School of Medicine cock et al., 1990). Whether the modified C terminus of processed ras mediates protein-phospholipid or pro-New York, New York 10016 tein-protein interactions or both is unknown. Whereas the prenyltransferases are soluble (Casey and Seabra, 1996), the activities of the enzymes that act Summary subsequent to prenyltransferase, including the prenyl-CAAX protease (Hancock et al., 1991a), pcCMT (Pillinger We show that Nras is transiently localized in the Golgi et al., 1994), and protein palmitoyltransferase (Kasinaprior to the plasma membrane (PM). Moreover, green than et al., 1990), are associated with membranes. Two fluorescent protein (GFP)-tagged Nras illuminated moof these three classes of enzymes, the prenyl-CAAX protetile, peri-Golgi vesicles, and prolonged BFA treatment ase and pcCMT, were recently characterized at the moblocked PM expression. GFP-Hras colocalized with lecular level, allowing unambiguous subcellular localiza-GFP-Nras, but GFP-Kras4B revealed less Golgi and no tion. Genetic screens of Saccharomyces cerevisiae vesicular fluorescence. Whereas a secondary memidentified genes for two metalloproteases with farnesylbrane targeting signal was required for PM expression, CAAX protease activity, STE24/AFC1 (Boyartchuk et al., the CAAX motif alone was necessary and sufficient to 1997; Fujimura-Kamada et al., 1997) and RCE1 (Boytarget proteins to the endomembrane where they were artchuk et al., 1997). Each gene predicted a polytopic methylated, a modification required for efficient memmembrane protein localized in the endoplasmic reticubrane association. Thus, prenylated CAAX proteins do lum (ER) (Schmidt et al., 1998). Similarly, both the S. not associate directly with the PM but instead associcerevisiae prenylcysteine carboxyl methyltransferase ate with the endomembrane and are subsequently Ste14p (Romano et al., 1998) and its human homolog transported to the PM, a process that requires a sec-pcCMT (Dai et al., 1998) are putative polytopic memondary targeting motif. brane proteins localized in the ER. Using enhanced green fluorescent protein (GFP)-tagged pcCMT, we
PLOS ONE, 2015
Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remai... more Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gainof-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At 20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.
Journal of leukocyte …, 1992
Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the... more Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP-3a, NP-4, and NP-5. The preprodefensins NP-3a, NP-4, and NP-5, like the previously characterized preprodefensins, lack consensus sequences for N-linked glycosylation, suggesting that defensins are targeted to lysosome-like granules by a mechanism not dependent on the mannose-6-phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.
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Papers by David Michaelson