Cdc42 controls spatial organization of autophagy in hematopoietic stem cells Free

Autophagy is a fundamental cellular process essential for hematopoietic stem cell (HSC) maintenance by removing damaged organelles/cellular components and preserving metabolic homeostasis. Disruption of this process has been causally associated with HSC exhaustion, impaired regenerative capacity, and aging (Ho et al., Nature 2017; Revuelta et al., Aging Cell 2017). While the core signaling pathways involved in autophagy initiation and flux are well defined, additional regulatory mechanisms, particularly those involved in autophagosome spatial organization, remain poorly understood. Cdc42, a Rho GTPase, is known to regulate cytoskeletal organization, cell polarity, and stem cell aging in HSCs (Florian et al., Cell Stem Cell 2012). We hypothesize that Cdc42 activity plays a role in autophagy regulation, particularly in interfacing cell organelle/autophagosome polarity and autophagy flux directionality.

To investigate this hypothesis, we generated GFP-LC3 autophagy reporter in Mx1-Cre; Cdc42^flox/floxmice for a hematopoietic-specific Cdc42 knockout model (Cdc42^flox/flox; Mx1-Cre; GFP-LC3), alongside littermate controls (Cdc42^flox/flox; GFP-LC3). Following pIpC induction, bone marrow cells were harvested and HSCs (Lin⁻Sca-1⁺c-Kit⁺CD135⁻CD48⁻CD150⁺) were analyzed under steady-state conditions for basal GFP-LC3 fluorescence; HSC cells were also treated ex vivo with bafilomycin A1 under starvation or rapamycin treatment to assess autophagy flux response. The autophagy-related cellular changes were comprehensively evaluated by flow cytometry, immunofluorescence microscopy, and Western blotting of key autophagy markers. At the steady state, Cdc42-deficient HSCs showed increased basal GFP-LC3 fluorescence intensity relative to controls, suggesting an autophagosome accumulation. Western blotting for key autophagosome biogenesis markers saw that LC3-II levels increased after bafilomycin A1 treatment in both genotypes, indicating ongoing autophagosome turnover. Interestingly, the LC3-II protein levels were comparable between Cdc42-deficient and control HSCs, and other autophagy markers, including phosphorylated ULK1, ATG5, and p62, were also comparable, suggesting that autophagosome initiation and biogenesis are largely preserved despite of Cdc42 loss. Notably, subsequent immunofluorescence analyses revealed a drastic spatial disorganization of cellular organelles in Cdc42-deficient HSCs. Unlike controls which displayed perinuclear LC3 clustering and strong co-localization with the LAMP1-positive lysosomes, Cdc42-deficient HSCs showed diffuse LC3 distribution, reduced LC3 co-localization with LAMP1, and a loss of polarity marked by disorganized tubulin, indicating that loss of Cdc42 disrupted spatial coordination required for proper autophagic vesicle trafficking and fusion with lysosomes. In addition, preliminary studies indicate that loss of Cdc42GAP, a negative regulator of Cdc42 activity, in a conditional Mx1-Cre; Cdc42GAP^flox/flox knockout mouse model, resulted in a reduced cell organelle polarity and disoriented autophagosome localization in HSCs, suggesting that hyperactivation of Cdc42 can also alter the directionality of autophagic vesicle trafficking in parallel to causing a loss of polarity as previously reported (Florian et al., Cell Stem Cell 2012). In aged HSCs from 18-25 months old mice that exhibited elevated Cdc42 activity similar to that seen in the Cdc42GAP KO mice, autophagy flux defects and LC3 mislocalization were also evident. A treatment of aged HSCs by the Cdc42-activity-specific inhibitor, CASIN, to reduce the elevated Cdc42 activity, was able to restore the LC3 polarity and autophagy flux defects, and rejuvenate the aged HSCs in effective engraftment and lineage balance.

Together, these findings demonstrate that Cdc42 activity is a key spatial regulator of autophagosome and autophagy flux in HSCs. Rather than affecting autophagy initiation or autophagosome assembly directly, Cdc42 coordinates autophagosome and lysosome positioning and fusion by maintaining cytoskeleton and organelle polarity, revealing a previously unrecognized regulatory axis linking polarity and autophagy. The studies provide new insights that modulating Cdc42 activity can revive HSC function under pathophysiologic conditions such as aging where cell polarity and normal autophagy flux are critical.