Research group Clemens Cabernard
Molecular and cellular mechanism of asymmetric stem cell division
Asymmetric cell division generates cellular diversity. Cell polarity, spindle orientation and cleavage furrow positioning are cellular mechanisms enabling cells to divide in a molecular and physical asymmetric manner. Stem cells in particular divide asymmetrically in order to self-renew the stem cell yet generate differentiating siblings. Many diseases such as breast cancer susceptibility, acute promyelocytic leukemia, the initiation of colon cancer but also the neurodevelopmental disorders lissencephaly or microcephaly are due to defective asymmetric stem cell division. Thus, understanding the cellular and molecular mechanisms of asymmetric cell division is important to increase our knowledge of basic stem cell biology.
We are using Drosophila melanogaster neuroblasts, the precursors of the fly’s central nervous system, to study the mechanism of (1) spindle orientation and (2) cleavage furrow positioning during asymmetric cell division. Neuroblasts are polarized cells and divide in a stem cell-like fashion, undergoing repeated self-renewing asymmetric divisions (Figure 1 and Movie 1). The mitotic spindle invariably orients itself along the neuroblast intrinsic apical-basal polarity axis and asymmetric cleavage furrow positioning results in a physical and molecular asymmetric cell division, generating a large self-renewed apical neuroblast and a smaller differentiating basal ganglion mother cell (GMC). Drosophila neuroblasts provide an ideal experimental system because precise genetic manipulations are possible and superb imaging properties are available.
Mechanism and function of spindle orientation during asymmetric cell division
Asymmetric cell division relies on the correct orientation of the mitotic spindle in relation to an internal or external polarity axis. In Drosophila neuroblasts, the mitotic spindle aligns itself along the intrinsic apical-basal polarity axis such that after division only the apical neuroblast inherits the Par proteins, whereas the Mira/Pros complex proteins segregate into the GMC (Figure 2 and Movie 2). Spindle orientation is controlled through a conserved protein complex consisting of the apically localized protein Partner of Inscuteable (Pins; LGN/AGS3 in vertebrates), the coiled-coil protein Mushroom body defect (Mud; NuMA in vertebrates) and the small G-protein Gαi. Mud is a key effector protein in spindle orientation and is providing a physical interaction between the mitotic spindle and the apical cortex.
We are using Drosophila larval neuroblasts to specifically address the following questions:
(1) How is centrosome positioning controlled, in order to establish a properly oriented metaphase spindle?
(2) How is the orientation of the metaphase spindle maintained?
(3) What are the key proteins involved in spindle orientation and what is their temporal and spatial requirement?
In order to answer these questions, we utilize precise and powerful genetic tools in combination with high temporal and spatial resolution live imaging (spinning disc). We further use immunoprecipitation mass spectrometry (IPMS) and forward genetics to identify novel proteins and genes involved in centrosome positioning and spindle orientation maintenance.
Cellular and molecular mechanism of cleavage furrow positioning
Asymmetric cell division can result in the formation of molecularly and physically distinct siblings. We are using Drosophila neuroblasts to investigate how cell size differences are generated. In particular, we are focusing on the cellular and molecular mechanism of cleavage furrow positioning. Until recently, it was widely believed that cleavage furrow positioning is solely dependent on cues delivered by the mitotic spindle. However, new results suggest that two cues are used for the correct positioning of the contractile ring: (1) a microtubule-dependent cue and (2) a polarity derived signal. The novel polarity-dependent cleavage furrow positioning pathway is utilizing the two conserved polarity components Pins and Discs large (Dlg) (Figure 3 and Movie 3).
We are investigating how cellular polarity is translated into asymmetric Myosin localization and ultimately, asymmetric cleavage furrow positioning. Furthermore, we are also testing the idea whether other polarized cell types utilize the “polarity-dependent” pathway to position the cleavage furrow. We are using forward and reverse genetics, live imaging with high temporal and spatial resolution and biochemistry in order to identify the cellular and molecular mechanism of cleavage furrow positioning in Drosophila neuroblasts.