Cdc42‐Borg4‐Septin7 axis regulates HSC polarity and function
2021; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês
10.15252/embr.202152931
ISSN1469-3178
AutoresRavinder Kandi, Katharina Senger, Ani Grigoryan, Karin Soller, Vadim Sakk, Tanja Schuster, Karina Eiwen, Manoj B. Menon, Matthias Gaestel, Yi Zheng, Maria Carolina Florian, Hartmut Geiger,
Tópico(s)DNA Repair Mechanisms
ResumoArticle18 October 2021Open Access Transparent process Cdc42-Borg4-Septin7 axis regulates HSC polarity and function Ravinder Kandi Ravinder Kandi Institute of Molecular Medicine, Ulm University, Ulm, Germany These authors contributed equally to this work Search for more papers by this author Katharina Senger Katharina Senger Institute of Molecular Medicine, Ulm University, Ulm, Germany These authors contributed equally to this work Search for more papers by this author Ani Grigoryan Ani Grigoryan orcid.org/0000-0003-2757-7021 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Karin Soller Karin Soller Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Vadim Sakk Vadim Sakk Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Tanja Schuster Tanja Schuster Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Karina Eiwen Karina Eiwen Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Manoj B Menon Manoj B Menon orcid.org/0000-0001-5859-0347 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India Search for more papers by this author Matthias Gaestel Matthias Gaestel orcid.org/0000-0002-4944-4652 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Yi Zheng Yi Zheng Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA Search for more papers by this author Maria Carolina Florian Maria Carolina Florian orcid.org/0000-0002-5791-1310 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Hartmut Geiger Corresponding Author Hartmut Geiger [email protected] orcid.org/0000-0002-5794-5430 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Ravinder Kandi Ravinder Kandi Institute of Molecular Medicine, Ulm University, Ulm, Germany These authors contributed equally to this work Search for more papers by this author Katharina Senger Katharina Senger Institute of Molecular Medicine, Ulm University, Ulm, Germany These authors contributed equally to this work Search for more papers by this author Ani Grigoryan Ani Grigoryan orcid.org/0000-0003-2757-7021 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Karin Soller Karin Soller Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Vadim Sakk Vadim Sakk Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Tanja Schuster Tanja Schuster Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Karina Eiwen Karina Eiwen Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Manoj B Menon Manoj B Menon orcid.org/0000-0001-5859-0347 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India Search for more papers by this author Matthias Gaestel Matthias Gaestel orcid.org/0000-0002-4944-4652 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Yi Zheng Yi Zheng Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA Search for more papers by this author Maria Carolina Florian Maria Carolina Florian orcid.org/0000-0002-5791-1310 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Hartmut Geiger Corresponding Author Hartmut Geiger [email protected] orcid.org/0000-0002-5794-5430 Institute of Molecular Medicine, Ulm University, Ulm, Germany Search for more papers by this author Author Information Ravinder Kandi1, Katharina Senger1, Ani Grigoryan1, Karin Soller1, Vadim Sakk1, Tanja Schuster1, Karina Eiwen1, Manoj B Menon2,3, Matthias Gaestel2, Yi Zheng4, Maria Carolina Florian1,5 and Hartmut Geiger *,1 1Institute of Molecular Medicine, Ulm University, Ulm, Germany 2Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany 3Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India 4Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA 5Present address: Program of Regenerative Medicine, IDIBELL, Barcelona, Spain *Corresponding author. Tel: +49 731 50 26700; Fax: +49 731 50 26710; E-mail: [email protected] EMBO Reports (2021)22:e52931https://doi.org/10.15252/embr.202152931 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid-primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42-Borg4-Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells. SYNOPSIS A Cdc42-Borg4-Septin7 axis regulates the intracellular distribution of polarity proteins in HSCs. Either Borg4 or septin7 are critical for HSC function upon stress, suggesting a role in compartmentalization that is lost upon aging due to elevated Cdc42 activity. Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate their polar distribution within HSCs. Lack of Borg4 or Septin7 in HSCs alters the distribution of Cdc42, likely by a feedback loop mechanism. HSCs devoid of Borg4 or Septin7 show reduced engraftment potential and hallmarks of aged HSCs. Introduction The function of hematopoietic stem cells (HSCs) decreases upon aging. This contributes, among others, to aging-associated immune remodeling (AAIR) with a reduced production of lymphoid cells (B cells and T cells) and erythroid cells in bone marrow (BM) as well as an increased myeloid output linked to aging-related myeloid malignancies (Rossi et al, 2008; Beerman et al, 2010; Geiger et al, 2013; Snoeck, 2013; Akunuru & Geiger, 2016). A prominent aging-related phenotype of aged HSCs is a reduced frequency of HSCs with a polar distribution of polarity proteins like Cdc42 or the cytoskeletal protein tubulin or the epigenetic marker histone 4 acetylated on lysine 16 (H4K16ac; Florian et al, 2012). Polarity within HSCs is tightly linked to the mode of symmetrical or asymmetrical division. Young (polar) HSCs divide more asymmetrically, while aged (apolar) HSCs divide more symmetrically (Florian et al, 2018). The aging-related changes in HSCs and hematopoiesis are caused by both cell intrinsic alterations in HSCs and extrinsic BM niche factors (Kamminga & de Haan, 2006; Geiger et al, 2007; Wang et al, 2011; Guidi et al, 2017; Saçma et al, 2019; Mejia-Ramirez & Florian, 2020). An increase in the activity of the small RhoGTPase Cdc42 in aged HSCs causes the reduced frequency of polar HSCs upon aging and is also causative for HSC aging (Florian et al, 2012; Grigoryan et al, 2018; Leins et al, 2018; Liu et al, 2019; Amoah et al, 2021). Pharmacological attenuation of the aging-related increase in the activity of Cdc42 by a specific small molecule inhibitor of Cdc42 activity termed CASIN resets the frequency of polar HSCs back to level reported for young HSCs and rejuvenates the function of chronologically aged HSC. Cdc42 activity is thus a critical regulator of HSC polarity and aging (Florian et al, 2020). While there is information on polarity regulation pathways orchestrated by Cdc42 activity in yeast (Okada et al, 2013; Chollet et al, 2020), the mechanisms by which Cdc42 controls polarity and especially how elevated activity of Cdc42 results in loss of polarity in HSCs are not understood. Ectopic expression of either a constitutively active form of Cdc42 or a dominant negative form of Cdc42 leads to redistribution of the borg family of Cdc42 effector proteins (also called Cdc42ep1-5) and subsequent loss of Septin filaments in cell lines (Joberty et al, 2001; Farrugia & Calvo, 2017). Septins are GTP-binding proteins that interact in stable stoichiometry within each other to form filaments that bind to the cell membrane, actin filaments, and microtubules. Septins are regarded as the fourth component of the cytoskeleton (Mostowy & Cossart, 2012). In yeast, Septins are recruited to the site of polarization by active Cdc42 while at the same time inhibiting Cdc42 activity in a negative feedback loop (Okada et al, 2013) In budding yeast gic-1, a functional homologue of mammalian Borg proteins, binds to cdc42-GTP which in turn leads to dissociation of gic1 from Septin filaments (Brown et al, 1997; Sadian et al, 2013, 1). Septins have gained recently more attention, also with respect to the hematopoietic system, like we and others have reported distinct roles for Septin6 (Senger et al, 2017) and Septin1 (Ni et al, 2019) in hematopoiesis, while the borg family of Cdc42 effector proteins still remains largely uncharacterized, particularly within the hematopoietic system. Here, we demonstrate that a Cdc42-Borg4-Septin7 axis regulates the intracellular distribution of polarity proteins in HSCs in response to changes in Cdc42 activity. Consequently, either Borg4 or Septin7 is critical for HSC function upon stress. Our results provide a novel scientific rationale for a role of Borgs and Septins in the compartmentalization of components within stem cells likely to be essential for stem cell function. Results Borg4 and Septin7 show a polar distribution in HSCs, which is regulated by Cdc42 activity There are 5 known distinct binders of Rho GTPases proteins (Borgs), also known as Cdc42ep4 1-5, which can serve as Cdc42 effector proteins. Borgs are able to bind septins via a conserved BD3 domain and interact with Cdc42 through a Cdc42/Rac interactive binding (CRIB) motif. Borgs play an important role in cytoskeletal rearrangement. They also regulate cell shape, filopodia formation, and cell migration and which are controlled by specific interaction with active Cdc42-GTP (Farrugia & Calvo, 2016). We first determined the level of expression of Borgs in HSCs. Borgs2–4 were expressed in LT-HSCs (Lin, c-Kit+, Sca-1+, flk2−, CD34− cells) (Fig 1A), whereas Borgs1 and 5 were absent or below the level of detection (Appendix Table S2). Expression of Borg2 and 3 was increased and Borg4 was decreased in aged (elevated level of Cdc42 activity) compared to young LT-HSCs. There are 13 distinct mammalian Septins. Septin2, 6, 7, 8, 9, 10, or 11 are thought to be ubiquitously expressed, while expression of Septin1, 3, 4, 5, 12, or 14 is usually restricted to distinct tissues or cell types. Septin1, 2, 6, 7, 8, 9, and 11 were indeed expressed in LT-HSC, while Septin3, 4, 5, 10, 12, and 14 were absent or below the level of detection (Appendix Table S1 and S2). The expression of Septin1, 2, 6, and 7 was decreased in aged LT-HSCs, while expression of Septin8 was increased upon aging (Fig 1B). The level of expression of Septin9 and 11 was similar in young and aged LT-HSCs. In summary, a distinct set of borgs and septins are expressed in LT-HSCs, and most borgs and septins that are expressed in HSCs show changes in expression in aged compared to young HSCs. Expression data on Septins and Borgs in murine HSCs and hematopoiesis from public data sets (bloodspot, https://servers.binf.ku.dk/bloodspot/, Figs EV2A–M and EV3A–F) are in general consistent with our expression data (Chambers et al, 2007). Figure 1. Borg4 and Septin7 show a polar distribution in HSCs which is regulated by Cdc42 activity A, B. Level of expression of Borgs and Septins in young and aged LT-HSCs. mRNA levels of Borgs and Septins were normalized to the level of expression of GAPDH and Borgs are shown relative to the level of Borg2 expression in young LT-HSCs. Septins are shown relative to the level of Septin7 expression in young LT-HSCs. At least three biological replicates (n = 3) are shown. Error bars indicate mean ± SEM, unpaired t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. C. Percentage of cells polarized for Septin7 among young, aged, and aged LT-HSCs treated with CASIN (5 µM). At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean ± SEM, unpaired t-test, **P < 0.01, ***P < 0.001. D, E. Representative immunofluorescence microscopy images of the distribution of Septin7 (red) or Borg3 or Borg4 (green) in young, aged, and aged LT-HSCs treated with CASIN (5 µM). Nuclei were stained with DAPI (blue), scale bar = 5 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Level of expression and polar distribution of Septins and Borgs in HSCs A–G. m-RNA expression levels of Septin1, 2, 6, 7, 8, 9, and 11 in young LT-HSCs, aged LT-HSCs and aged LT-HSCs treated with CASIN 5 µM normalized to GAPDH and shown relative to respective septins in young LT-HSCs, at least three biological replicates (n = 3). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. H–J. Expression levels of Borg2, Borg3, and Borg4 in young, aged, and aged LT-HSCs treated with CASIN 5 µM normalized to GAPDH and shown relative to respective borgs in young LT-HSCs. At least three biological replicates (n = 3) are shown. Error bars indicate mean ± SEM; ***P < 0.001, ****P < 0.0001. K. Percentage of cells polarized for Septin2 and Septin6 in young, aged, and aged LT-HSCs treated with CASIN 5 µM. Each sample cell was analyzed and scored for Septin-2 and Septin6 polarity distribution. Two biological repeats (n = 2) with at least 20 cells per repeat are shown. Error bars indicate mean ± SD. L, M. Representative images of Septin2, Septin6 (red), and tubulin (green) distribution in young, aged, and aged LT-HSCs treated with 5 µM CASIN by immunofluorescence microscopy. Nuclei were stained with DAPI (blue); scale bar = 5 µm. N. Representative images of the distribution of Cdc42-GTP (orange), Borg4 (red), and Septin7 (green) in young and aged LT-HSCs (by immunofluorescence microscopy). Nuclei were stained with DAPI (blue); scale bar = 5 µm. O. Percentage of cells polarized for Cdc42-GTP, Borg4, and Septin7 in young or aged LT-HSCs, n = 3. Error bars indicate mean ± SD, *P < 0.05 (two-way ANOVA). P. Quantification of the co-localization of Cdc42-GTP, Borg4 or Septin7 proteins in LT-HSCs. Error bars indicate mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Expression pattern of septins in hematopoiesis A. Expression of septins in LT-HSCs. n = 4 data points, error bars indicate mean ± SD. B–M. Expression of Septins in stem cells and hematopoiesis. The x-axis indicates stem and progenitor cells names; the y-axis indicates Log2 expression values. Data and graphs from blood spot (https://servers.binf.ku.dk/bloodspot/). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Expression pattern of Cdc42eps (Borgs) in hematopoiesis A. Expression of Cdc42eps (Borgs) in LT-HSCs. n = 4 data points, error bars indicate mean ± SD. B–F. Expression of Cdc42eps (Borgs) in stem cells and hematopoiesis. The x-axis indicates stem and progenitor cells names; the y-axis indicates Log2 expression values. (G) Schematic representation of the proximity ligation assay reaction for Cdc42-Borg4 and Borg4-Septin7 interactions. H. Representative confocal 3D image of LT-HSC from Cdc42 KO mice showing Cdc42-Borg4 interaction as a control. Nuclei were stained with DAPI; scale bar = 5 µm. Data and graphs from blood spot (https://servers.binf.ku.dk/bloodspot/). Download figure Download PowerPoint Septins function in a physiological manner to align with scaffolds (tubulin, actin, and vimentin) to allow for compartmentalization. In yeast, Septins control the diffusion of specific proteins between mother and bud cells, regulating cellular aging (Takizawa et al, 2000; Shcheprova et al, 2008). We therefore investigated whether the spatial distribution of Septin proteins is distinct in young and aged LT-HSCs using immunofluorescence analyses, and if so, whether changes in the distribution are a consequence of the elevated level of Cdc42 activity in aged LT-HSCs. The core Septin filament structure consists of Septin2, 6, and 7 (Low & Macara, 2006; Sirajuddin et al, 2007). We therefore focused first on the localization of Septin2, 6, and 7 in HSCs. The distribution of Septin7 was polarized in about 50% of young but only in about 30% of aged LT-HSCs (Figs 1C and D, and EV1O), while only a very low frequency of LT-HSC (young or aged) were polar for either Septin2 or Septin6 (Fig EV1K–M). Aged HSCs possess an elevated activity of Cdc42 (more Cdc42-GTP) in comparison with young HSCs. The level of Cdc42 activity has been shown to control the spatial distribution of Septins via Borg effector proteins in both yeast and mammalian cell lines (Joberty et al, 2001). The distribution of Septins in HSCs might therefore be regulated by the interaction of Septins with the Borg family of Cdc42 effector proteins (Sheffield et al, 2003; Farrugia & Calvo, 2016). We first tested whether a reduction of the elevated activity of Cdc42 in aged HSCs to the level reported for young HSCs with a specific inhibitor of Cdc42 activity termed CASIN (Florian et al, 2012; Grigoryan et al, 2018; Leins et al, 2018; Liu et al, 2019; Amoah et al, 2021) might influence distribution of Septin2, 6 or 7 in aged LT-HSCs. Inhibition of Cdc42 activity increased the frequency of aged LT-HSCs polar for Septin7 to the frequency reported for young LT-HSCs (Fig 1C and D) while the distribution of Septin2 and 6 was not affected by CASIN (Fig EV1K–M). We also tested whether Borgs expressed in LT-HSCs and for which antibodies were available (Borg3 (Cdc42ep5) and Borg4 (Cdc42ep4) might show co-distribution with septins. Borg4 co-localized with Septin7 in young and aged, CASIN treated LT-HSCs repolarized for Septin7, but not in aged LT-HSCs (Fig 1D), while Borg3 did not co-localize with Septin7 (Fig 1E). In summary, our data support that the activity of Cdc42 regulates the subcellular compartmentalization of HSCs in very specific and targeted manner, due to the fact that the reduction of Cdc42 activity restored the polarity of Septin7 and Borg4, but not affecting Septin2, 6, and Borg3 distribution. We also investigated whether the reduction of Cdc42 activity in aged HSCs by CASIN affected the expression of Septins and Borgs in LT-HSCs to test for a role of Cdc42 activity in regulating the expression of septins and borgs in addition to controlling their distribution. Interestingly, the level of expression Septin1, 2, 6, 7, 8, 9, and 11 was increased in CASIN treated aged HSCs, while the expression of septin8 was decreased and thus also more similar to the level in young LT-HSCs (Fig EV1A–G). Similarly, the level of expression of Borg2 and 4 in aged LT-HSCs was more similar to the level in young LT-HSCs upon reduction of Cdc42 activity by CASIN, while the level of Borg3 even further increased in aged LT-HSCs upon inhibition of Cdc42 activity (Fig EV1H–J). Analysis of the spatial distribution of CdC42GTP along with Septin7 and Borg4 identified that these proteins can indeed co-localize in both young and aged LT-HSCs and confirmed an increase in the level of Cdc42-GTP in aged LT-HSCs (Fig EV1N–P). In summary, our data support an influence of Cdc42 activity on the level of expression of distinct Borgs and Septins, but more importantly on the spatial polar distribution of especially Borg4 and Septin7 in LT-HSCs. Interestingly, Borg4 seems to specifically interact with Cdc42 but not to other small RhoGTPases like RhoA or Rac1 (Joberty et al, 1999, 10; Hirsch et al, 2001). On the other hand, Septin7 has a particular role in the Septin family due to the fact that it is the only member of its subgroup that cannot be replaced by any other septin in a hexameric or octameric septin assembly (Kremer et al, 2005; Tooley et al, 2009; Kim et al, 2011, 9). The Cdc42-Borg4-Septin7 interaction in HSCs is regulated by the level of Cdc42 activity It has been proposed that Cdc42-GTP, but not GDP, binds to borg proteins to ideally position them within the cell while a switch to Cdc42-GDP releases borgs from Cdc42 to allow for interactions with for example Septins to stabilize Septin networks (Farrugia & Calvo, 2016). We tested such a likely direct physical interaction between Cdc42-Borg4 due to their co-distribution in LT-HSCs by a proximity ligation assay (PLA) (Fig 2A). A positive fluorescence signal in a PLA assay indicates a proximity two distinct proteins of at least less than 40 nm between the epitopes which usually equals to physical interaction (Fig EV3G). We observed multiple spots of interactions between Cdc42 and Borg4 in young HSCs, the level of which was elevated in aged LT-HSCs. Aged LT-HSCs in which Cdc42 activity was adjusted by CASIN showed again a level of reduced interaction similar to young LT-HSCs. This implies that the level of Cdc42 activity regulates the level of the Borg4-Cdc42 interaction, with elevated levels of Cdc42-GTP in aged LT-HSCs resulting in enhanced binding of Borg4 to Cdc42 (Fig 2A and B). Similarly, we tested for a physical interaction of Borg4 with Septin7 (Fig 2C and D). There was more interaction in young and aged LT-HSC treated with CASIN, and less in aged LT-HSCs (high Cdc42GTP), implying that Borg4 is indeed more likely to be released from Cdc42GDP to then interact with Septin7 (Fig 2B and D). In case of the Cdc42 and Borg4 interaction, we used Cdc42 knockout cells as a negative control which indeed delivered only minor background signal (Fig EV3 H). The attenuation of the elevated activity of Cdc42 in aged HSCs to the level found in young HSCs regulated the extent of the interaction between Cdc42 and Borg4 and in turn between Borg4 and Septin7. Among the Borgs and Septins expressed in LT-HSCs, Borg4 and Septin7 thus specifically build a critical "effector cascade of Cdc42 activity" to confer outcomes on LT-HSCs (like level of the polar distribution of polarity proteins) in response to changes Cdc42 activity in LT-HSCs. Figure 2. The physical interaction between Cdc42-Borg4-Septin7 is regulated by the activity of Cdc42 Representative immunofluorescence confocal microscopy images of young, aged, and aged + CASIN (5 µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and Borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar = 5 µm. Quantification of the level of fluorescent signal in young, aged, and aged + CASIN (5 µM) treated LT-HSCs to quantify the Cdc42-Borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n = at least 17 cells per condition, error bars indicate mean ± SEM, unpaired t-test, ****P < 0.0001. Representative immunofluorescence confocal microscopy images of young, aged, and aged + CASIN (5 µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Borg4 and Septin7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar = 5 µm. Quantification of the level of fluorescent signal in young, aged, and aged + CASIN (5 µM) treated LT-HSCs to quantify the Borg4-Septin7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n = at least 17 cells per condition; error bars indicate mean ± SEM, unpaired t-test, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Borg4 or Septin7 regulate the distribution of polarity proteins in HSCs A general genetic ablation of Septin7 is embryonic lethal, while general genetic ablation of Borg4 results in neurological defects (Menon et al, 2014; Ageta-Ishihara et al, 2015; Abbey et al, 2016). To further investigate the role of Borg4 and Septin7 for Cdc42-driven phenotypes like polarity in specifically HSCs and hematopoietic cells, we used a vav-1 driven cre-recombinase to delete Borg4 or Septin7 in hematopoietic cells of Borg4flox/flox or Septin7flox/flox animals (leading to Borg4Δ/Δ or Septin7Δ/Δ) (Menon et al, 2014; Ageta-Ishihara et al, 2015). Deletion of Borg4 or Septin7 in hematopoietic cells, including LT-HSCs from Borg4Δ/Δ or Septin7Δ/Δ animals, was confirmed by PCR, quantitative RT-PCR or immunofluorescence (Fig EV4A–D). The absence of Borg4 from LT-HSCs resulted in a reduced frequency of LT-HSC polar for the distribution of Septin7 and tubulin but interestingly also for Cdc42 itself (Fig 3A–C), while the frequency of cells polar for the epigenetic polarity marker histone 4 acetylated on lysine 16 (H4K16ac) was only slightly affected by the lack of Borg4 (Fig EV4F and I). The absence of Septin7 from LT-HSCs resulted in a reduced frequency of LT-HSC polar for the distribution of Borg4, tubulin, and, similar to the borg4Δ/Δ LT-HSCs, also for Cdc42 (Fig 3D–F) and for H4K16ac (Fig EV4E and H). This implies a feedback loop in which changes in the Septin7 localization and thus likely a disturbed Septin network will in return influence the localization of Cdc42 as well as Borg4. Such a general feedback loop between Cdc42 localization and Septins has been previously described in yeast (Okada et al, 2013) but not yet for stem or even for mammalian cells. Interestingly, the distribution of either Septin2, or Septin6, which are, similar to Septin7, components of the core Septin filament complex, is not affected by a lack of Borg4 (Fig EV4G and J) nor is the distribution of Crumbs3, another marker of polarity in epithelial cells (Fig EV4K and L). This implies that the positions of core complex Septins within HSCs are not synchronized. Click here to expand this figure. Figure EV4. Distribution of polarity proteins in Borg4Δ/Δ or Septin7Δ/Δ HSCs A. QRT-PCR analysis of Borg4 mRNA in low-density bone marrow cells isolated from Borg4fl/fl and Borg4Δ/Δ mice (n = 3, three biological replicates). ****P < 0.0001. Unpaired t-test, error bars indicate mean with SD. B. PCR amplification of genomic DNA from lineage negative cells of Borg4fl/fl and Borg4Δ/Δ Here, we used two sets of primers to distinguish Borg4fl/fl (with primer a + b: 580 bp) and Borg4Δ/Δ (primer a + c: 490 bp). For the amplification of the cre allele (375 bp), another set of primers was used. C. Immunofluorescence staining of Septin7(red) in LT-HSCs of Septin7fl/fl and Septin7Δ/Δ mice. Scale bar = 5 µm. D. Septin7 knock-out confirmation by PCR amplification of genomic DNA in lineage negative cells using primers P1, P2, and P3. E, F. Representative images of H4K16ac (red) and tubulin (green) distribution in Borg4fl/fl and Borg4Δ/Δ; Septin7fl/fl and Septin7Δ/Δ LT-HSCs by immunofluorescence microscopy. Nuclei were stained with DAPI (blue), scale bar = 5 µm. G. Representative images of Septin2 (cyan) and Septin6 (orange) distribution in Borg4fl/fl and Borg4Δ/Δ. Scale bar = 5 µm. H, I. Percentage of cells polarized for H4K16ac in Borg4fl/fl and Borg4Δ/Δ; Septin7fl/fl or Septin7Δ/Δ LT-HSCs. At least three biological replicates (n = 3) are shown. Error bars indicate mean ± SD, *P < 0.05, unpaired t-test. J. Percentage of cells polarized for Septin2 and Septin6 in Borg4fl/fl and Borg4Δ/Δ LT-HSCs. At least three biological replicates (n = 3) are shown. Error bars indicate mean ± SD. K. Representative images of Crumbs3 (purple) distribution in Borg4fl/fl or Borg4Δ/Δ LT-HSCs by immunofluorescence microscopy. Nuclei were stained with DAPI (blue), scale bar = 5 µm. L. Percentage of cells polarized for Crumbs3 in Borg4fl/fl or Borg4Δ/Δ LT-HSCs. At least, three biological replicates (n = 3) are shown. Error bars indicate mean ± SD. Download figure Download PowerPoint Figure 3. Borg4 or Septin7 regulates the distribution of polarity proteins in HSCs A, B. Representative immunofluorescence microscopy images of the distribution of Septin7 (red), Cdc42 (red), and tubulin (green) in LT-HSCs from Borg4fl/fl or Borg4Δ/Δ mice. Nuclei were stained with DAPI (blue), scale bar = 5 µm. C. Percentage of cells with a polar distribution of Septin7, Cdc42 or tubulin in Borg4fl/fl or borg4Δ/Δ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean ± SEM, two-way ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05. D, E. Representative immunofluorescence microscopy imag
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