The cell is the fundamental unit of life, composed of smaller, non-living components. This raises a key question: what truly gives rise to life? This thesis explores cytoskeletal organization and dynamics, focusing on microtubules and spindle positioning, with the broader aim of reconstituting these processes in synthetic cells.

Encapsulation of biological components is central to synthetic cell research. We evaluated different methods to create cell-like compartments, finding that while droplets are easy to work with, cDICE offers greater flexibility for functional encapsulation. Using high-speed imaging, we studied GUV formation in cDICE and discovered a size-selective crossing of droplets at the interface. We also found that proteins in the inner solution affect GUV formation by increasing viscosity and altering lipid adsorption.

Tubulin, an essential protein in cells, remains difficult to work with in vitro. Using different encapsulation methods, we observed that tubulin influences the stability of lipid bilayers, and using a membrane interaction assay, we found that it can even disrupt the membranes.

To increase biological relevance, we combined major cellular components like microtubule asters with an actin cortex or a nucleus mimic, and explored external tools for spatiotemporal control. We successfully assembled a mitotic spindle-like organization in droplets. We incorporated an optogenetic switch to control dynein in order to achieve asymmetric spindle positioning, inspired by the first cell division of the C. elegans embryo. However, light-induced transport remains limited.

Finally, we adapted the bacterial ParMRC DNA segregation system for synthetic cell, with light-regulated control via iLID. Although individual components react to light activation, further optimization is needed to make the full system responsive.

These reconstitutions provide insight into fundamental mechanisms of spindle positioning and are basic steps toward building a minimal synthetic spindle.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

Background: Rural India has a severe shortage of human resources for health (HRH). The National Rural Health Mission (NRHM) deploys HRH in the rural public health system to tackle shortages. Sanctioning under NRHM does not account for workload resulting in inadequate and inequitable HRH allocation. The Workforce Indicators of Staffing Needs (WISN) approach can identify shortages and inform appropriate sanctioning norms. India currently lacks nationally relevant WISN estimates. We used existing data and modelling techniques to synthesize such estimates. Methods: We conducted a retrospective analysis of existing survey data for 93 facilities from 5 states over 8 years to create WISN calculations for HRH cadres at primary and community health centres (PHCs and CHCs) in rural areas. We modelled nationally representative average WISN-based requirements for specialist doctors at CHCs, general doctors and nurses at PHCs and CHCs. For 2019, we calculated national and state-level overall and per-centre WISN differences and ratios to depict shortage and workload pressure. We checked correlations between WISN ratios for cadres at a given centre-type to assess joint workload pressure. We evaluated the gaps between WISN-based requirements and sanctioned posts to investigate suboptimal sanctioning through concordance analysis and difference comparisons. Results: In 2019, at the national-level, WISN differences depicted workforce shortages for all considered HRH cadres. WISN ratios showed that nurses at PHCs and CHCs, and all specialist doctors at CHCs had very high workload pressure. States with more workload on PHC-doctors also had more workload on PHC-nurses depicting an augmenting or compounding effect on workload pressure across cadres. A similar result was seen for CHC-specialist pairs—physicians and surgeons, physicians and paediatricians, and paediatricians and obstetricians–gynaecologists. We found poor concordance between current sanctioning norms and WISN-based requirements with all cadres facing under-sanctioning. We also present across-state variations in workforce problems, workload pressure and sanctioning problems. Conclusion: We demonstrate the use of WISN calculations based on available data and modelling techniques for national-level estimation. Our findings suggest prioritising nurses and specialists in the rural public health system and updating the existing sanctioning norms based on workload assessments. Workload-based rural HRH deployment can ensure adequate availability and optimal distribution.