Investigating the mechanisms of lysosome segregation and cell fate regulation in dividing hematopoietic stem cells

PROJECT SUMMARY Hematopoietic stem cell (HSC) transplantation is the only curative therapy for many diseases, but low HSC numbers and their tendency to differentiate in vitro are limiting factors for the more widespread application of this life-saving therapy. Knowing how HSCs decide between self-renewal and differentiation is critical to develop

efficient culture systems that manipulate HSC fate decisions to either expand or differentiate HSCs into specific mature blood cells for transplantation. However, HSC decision-making processes are incompletely understood and our ability to expand HSCs is limited. HSC self-renewal and differentiation are regulated by the symmetric

and asymmetric segregation of cell fate determinants (CFD) during division. After asymmetric segregation of CFDs, only one daughter retains HSC properties, while symmetric segregation of CFDs can create two HSCs. An exciting prediction of this model is, that factors promoting symmetric segregation of CFD during division

expand HSCs. However, identifying these factors has been hampered because of major technical challenges including: 1) HSCs are rare and divide infrequently, making transient events like divisions hard to capture in fixed samples, and 2) cell fate acquisition after HSC division takes many days. Understanding how the segregation of

CFD influences cell fate acquisition thus requires precise quantifications of living HSCs over many days with single-cell resolution. To address this challenge, we developed novel long-term live-cell imaging tools, that allow us to track and quantify single HSC behaviors and marker expression over several weeks and cell generations.

This allowed us to visualize for the first time that lysosomes are asymmetrically segregated during HSC divisions and act as CFD to instruct later changes in metabolism and differentiation in HSC daughter cells. Here, we propose to leverage this novel technology to identify factors regulating lysosome segregation and to investigate

how RhoGTPases, which are known polarity proteins in invertebrates, regulate lysosome segregation, and HSC self-renewal and differentiation. Studies of non-dividing cells showed that Tubulin, Actin and Vimentin filaments of the cytoskeleton regulate lysosome motility. However, which of these filaments regulates lysosome motility

during HSC division remains unclear. To address this knowledge gap, we will study lysosomal motility and segregation in dividing HSCs using our live-cell imaging tools. We will assess the polarization and colocalization of lysosomes with Tubulin, Actin and Vimentin filaments and functionally interrogate how pharmacological and

genetic manipulation of known cytoskeletal transport mechanisms and polarity regulators, such as CDC42, affect lysosomal segregation and daughter cell fate acquisition. Our preliminary data, show that lysosome segregation is regulated via ARP2/3-mediated actin nucleation, which lies downstream of the Integrin/Focal Adhesion Kinase

(FAK). We also provide evidence that the RhoGTPases CDC42 and RAC have opposing roles and act as negative and positive regulators of asymmetric lysosome segregation, respectively. Based on these data, we hypothesize that HSCs regulate lysosome segregation through the Integrin-FAK-ARP2/3 pathway.