Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

109

BSTRACTThe midbody remnant (MBR) that is generated after cytokinetic abscission has recently attracted a lot of attention, because it might have crucial consequences for cell differentiation and tumorigenesis in mammalian cells. In these cells, it has been reported that the MBR is either released into the extracellular medium or retracted into one of the two daughter cells where it can be degraded by autophagy. Here, we describe a major alternative pathway in a variety of human and mouse immortalized cells, cancer cells and primary stem cells. Using correlative light and scanning electron microscopy and quantitative assays, we found that sequential abscissions on both sides of the midbody generate free MBRs, which are tightly associated with the cell surface through a Ca 2+ /Mg 2+ -dependent receptor. Surprisingly, MBRs move over the cell surface for several hours, before being eventually engulfed by an actin-dependent phagocytosis-like mechanism. Mathematical modeling combined with experimentation further demonstrates that lysosomal activities fully account for the clearance of MBRs after engulfment. This study changes our understanding of how MBRs are inherited and degraded in mammalian cells and suggests a mechanism by which MBRs might signal over long distances between cells.

Section: Resultsmentioning

confidence: 99%

Midbody remnant engulfment after cytokinesis abscission in mammalian cells

Crowell

Gaffuri

Gayraud-Morel

et al. 2014

109

“…Arf6 localizes to the midbody during the late stages of abscission, through interaction with mitosis kinesin-like protein 1 (MKLP1). The Arf6-MKLP1 complex links the plasma membrane to the microtubules running through the midbody, a step required for cell separation (Makyio et al, 2012). These functions of Arf6 are probably not essential to cytokinesis in all cells, as Arf6-knockout mice are viable at birth (Suzuki et al, 2006) and a Drosophila Arf6 2/2 mutant has cytokinesis defects only in spermatocytes (Dyer et al, 2007).…”

Section: Arf Function In Mitosismentioning

confidence: 99%

Arfs at a Glance

Jackson

Bouvet

2014

119

117

The Arf small G proteins regulate protein and lipid trafficking in eukaryotic cells through a regulated cycle of GTP binding and hydrolysis. In their GTP-bound form, Arf proteins recruit a specific set of protein effectors to the membrane surface. These effectors function in vesicle formation and tethering, non-vesicular lipid transport and cytoskeletal regulation. Beyond fundamental membrane trafficking roles, Arf proteins also regulate mitosis, plasma membrane signaling, cilary trafficking and lipid droplet function. Tight spatial and temporal regulation of the relatively small number of Arf proteins is achieved by their guanine nucleotideexchange factors (GEFs) and GTPase-activating proteins (GAPs), which catalyze GTP binding and hydrolysis, respectively. A unifying function of Arf proteins, performed in conjunction with their regulators and effectors, is sensing, modulating and transporting the lipids that make up cellular membranes. In this Cell Science at a Glance article and the accompanying poster, we discuss the unique features of Arf small G proteins, their functions in vesicular and lipid trafficking in cells, and how these functions are modulated by their regulators, the GEFs and GAPs. We also discuss how these Arf functions are subverted by human pathogens and disease states.

“…The expression vector for C-terminally EGFP-tagged SCAP was constructed by subcloning a cDNA fragment containing the entire coding sequence of human SCAP into pEGFP-N1. The expression vector for Nterminally mRFP-tagged SREBP1c was constructed by subcloning a cDNA fragment containing the entire coding sequence of human SREBP1c into the pcDNA3-Kozac-mRFP-C vector (Makyio et al, 2012). The R503A mutation of SREBP1c was introduced into the SREBP1c cDNA using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies).…”

Section: Recombinant Plasmids and Virusesmentioning

confidence: 99%

COPI-mediated retrieval of SCAP is critical for regulating lipogenesis under basal and sterol-deficient conditions

Takashima

Saitoh

Funabashi

et al. 2015

Self Cite

Retrograde trafficking from the Golgi complex to endoplasmic reticulum (ER) through COPI-coated vesicles has been implicated in lipid homeostasis. Here, we find that a block in COPI-dependent retrograde trafficking promotes processing and nuclear translocation of sterol regulatory element binding proteins (SREBPs), and upregulates the expression of downstream genes that are involved in lipid biosynthesis. This elevation in SREBP processing and activation is not caused by mislocalization of S1P or S2P (also known as MBTPS1 and MBTPS2, respectively), two Golgi-resident endoproteases that are involved in SREBP processing, but instead by increased Golgi residence of SREBPs, leading to their increased susceptibility to processing by the endoproteases. Analyses using a processing-defective SREBP mutant suggest that a fraction of SREBP molecules undergo basal cycling between the ER and Golgi in complex with SREBP cleavage-activating protein (SCAP). Furthermore, we show that SCAP alone is retrieved from the Golgi and moves to the ER after processing of SREBP under steroldeficient conditions. Thus, our observations indicate that COPImediated retrograde trafficking is crucial for preventing unnecessary SREBP activation, by retrieving the small amounts of SCAP-SREBP complex that escape from the sterol-regulated ER retention machinery, as well as for the reuse of SCAP.