Biology subjects

Sutanto, R.

Publications and source records attributed to Sutanto, R..

4 recordsLinked to original sources

bioRxiv2023

The oncogene cyclin D1 promotes bipolar spindle integrity under compressive force

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

cell biology↗

bioRxiv2022

Kinetochore-fiber lengths are maintained locally but coordinated globally by poles in the mammalian spindle

At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the poles contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.

cell biology↗

bioRxiv2021

The Astrin/SKAP Complex Reduces Friction at the Kinetochore-Microtubule Interface

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation, but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens movement and decreases coordination of metaphase sister kinetochores, and increases tension between them. Using laser ablation to isolate kinetochores bound to polymerizing vs depolymerizing microtubules, we show that without SKAP kinetochores move slower on both polymerizing and depolymerizing microtubules, and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to previously described kinetochore proteins that increase grip on microtubules under force, Astrin-SKAP reduces grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

cell biology↗

bioRxiv2021

Stage-specific, morphological and molecular markers of encystation in Giardia lamblia

Differentiation into environmentally resistant cysts is required for transmission of the ubiquitous intestinal parasite Giardia lamblia. Encystation in Giardia requires the production, processing and transport of Cyst Wall Proteins (CWPs) in developmentally-induced, Golgi-like, Encystation Specific Vesicles (ESVs). Progress through this trafficking pathway can be followed by tracking CWP localization over time. However, there is no recognized system to distinguish the advancing stages of this process which can complete at variable rates depending how encystation is induced. Here we propose a staging system for encysting Giardia based on the morphology of CWP1-stained ESVs. We demonstrate the molecular distinctiveness of maturing ESVs at these stages by following GlRab GTPases through encystation. Previously, we established that Giardias sole Rho family GTPase, GlRac, associates with ESVs and has a role in regulating their maturation and the secretion of their cargo. As a proof of principle, we delineate the relationship between GlRac and ESV stages. Through proteomic studies, we identify putative interactors of GlRac that could be used as additional stage-specific ESV markers. This staging system provides a common descriptor of ESV maturation regardless of the source of encysting cells. Furthermore, the identified set of molecular markers for ESV stages will be a powerful tool for characterizing trafficking mutants that impair ESV maturation and morphology. ImportanceGiardiasis is a diarrheal disease that affects 280 million people worldwide. It is caused by Giardia lamblia, a protozoan parasite which rely on differentiating from host-dwelling trophozoites to environmentally-resistant cysts for transmission and survival. This encystation process requires the transport of Cyst Wall Proteins (1-3) within membrane-bound compartments called Encystation Specific Vesicles (ESV) from the endoplasmic reticulum to the surface of the cell. The whole process takes 24 hours to complete and these compartments are the only recognizable equivalent of Golgi apparatus in this minimalistic organism. Progress of this trafficking pathway can be followed by localizing Cyst Wall Protein 1 over time post induction of encystation but this can be ambiguous when specific molecular events need to be specified. Here we propose a staging system that is based on ESV morphology changes by capitalizing on the secretory/processing events we already know they represent. We validate the molecular distinctiveness of these stages by following Giardia Rabs through the pathway and characterize putative interactors of an established regulator of encystation, GlRac, to provide additional stage-specific molecular markers. This staging system will provide a definitive, yet adaptable, framework to map out functions of yet-to-be discovered players of this important pathway.

cell biology↗