A New Mechanism of the Polar Distribution of Sodium and Potassium ATPase During Nematode Sperm Activation

On May 28, 2021, Developmental Cell published an online study titled "Membrane-contact site-dependent cholesterol transport regulates Na ⁺ /K ⁺ -ATPase polarization and spermiogenesis in Caenorhabditis elegans " from the Miao Long research group of the Institute of Biophysics, Chinese Academy of Sciences .

This paper reveals the molecular mechanism of the non-vesicular reverse transport of cholesterol based on membrane contact sites, which regulates the polarity distribution of sodium and potassium ATPase during the activation of nematode spermatozoa, and identifies two specific expressions of spermatogenesis Sodium-potassium ATPase encodes genes nkb-2 (regulatory subunit) and capt-4 (functional subunit) and explores the regulation mechanism of sodium-potassium ATPase ion pump maintaining the ion homeostasis in sperm cells and affecting sperm activation and movement

.

The establishment and maintenance of cell polarity is necessary for many biological processes such as cell growth, asymmetric cell division, cell directional migration, etc.

It is the selective distribution of cell membrane proteins and related signal molecules, intracellular cytoskeleton and organelles in specific areas As a result, its polarization state changes with changes in cell function, which is the basis for cell morphological diversity and plasticity.

At present, the molecular mechanism that regulates the establishment and maintenance of cell polarity is still unclear

.

The spermatogenesis of C.elegans , a model organism, has been studied for a long time.

After two meiosis, haploid sperm shed from the remnant body by budding.

The cell contains a nucleus, mitochondria, and a specialization.

The membrane organelles (membranous organelle, MO), (see ultrastructure diagram); while the ribosomes, endoplasmic reticulum, Golgi apparatus and other organelles are retained in the residual body and then degraded

.

The haploid sperm that completes meiosis is similar to mammalian sperm, and also requires a mature process (called sperm capacitation in mammals, sperm activation in nematodes) to develop into a function of motility and fertilization Sex sperm, in this process, the cell receives the activation signal to establish polarity, grow pseudopodia, MO and plasma membrane fusion to maintain the sperm polarity state, is a good model for studying the establishment and maintenance of motor cell polarity

.

 

C.elegans nematode sperm cell ultrastructure

Miao Long’s research group used C.

elegans as the material to study the regulation mechanism of sperm activation.

This study identified two spermatogenesis-specific genes nkb-2 and capt-4 , which code for the regulation of sodium-potassium ATPase ion pump (NKA).

Subunits and functional subunits, NKA is evenly distributed in the plasma membrane in resting sperm cells.

When the sperm cells receive activation signals and start to activate, the cells grow acicular pseudopods and initiate the establishment of polarity, and the acicular pseudopods fuse into a sheet At the same time as the pseudopodia, NKA gathers in the direction of the cell body.

As the MO fuses with the plasma membrane, NKA disappears from the front of the pseudopodia and sinks into the MO fusion fossa, and the sperm gains motility within 5 minutes (see video)
.
Mutations in any of the NKA subunits will increase the concentration of sodium ions in sperm cells, increase the membrane potential, abnormal aggregation of the major sperm protein (MSP), and greatly reduce the activation of sperm motility, resulting in nematode infertility
.
 

 NKA (GFP::NKB-2, green) dynamic polarity distribution during the activation of nematode spermatozoa, red (FM 4-64) marks the plasma membrane
.

At the same time, the study focused on the analysis of the regulation mechanism of the polarity distribution of NKA during sperm activation, and found that there is a co-transport phenomenon between NKA and plasma membrane cholesterol.
The electron microscopy ultrastructure of sperm cells shows the difference between the MO head of the intracellular membrane organelle and the plasma membrane.
A stable membrane contact site is formed between them, and the key proteins that regulate cholesterol/PI4P transport in other tissues and cells, such as OSBP/OBR-4, VAP/VPR-1, SAC-1, etc., are all expressed in nematode sperm and localized in MO In the head, the inositol 5-phosphatase CIL-1, which uses PI(4,5)P2 as a substrate to produce PI4P, is also necessary for the activation of nematode sperm (Bae et al., 2009).
The mutation of CIL-1 can As a result, NKA cannot be transported to the MO fusion pore when sperm is activated, suggesting that the decrease of PI4P at the membrane contact site inhibits the transport of ion pump proteins, which in turn affects the maintenance of cell polarity and the acquisition of cell motility
.

This work reveals that the conversion of membrane phospholipid components PI(4,5)P2 and PI4P in sperm cells affects the reverse transport of plasma membrane cholesterol during sperm activation and movement, thereby regulating the polarity distribution of the plasma membrane sodium potassium ATPase ion pump; and sodium potassium The ion balance regulated by the ATPase ion pump is necessary for sperm activation and fertilization.
It is explained that the sodium-potassium ATPase ion pump located in the plasma membrane breaks the symmetry and performs polar positioning during sperm activation and regulates the intracellular Ion balance maintains the molecular mechanism of sperm motility and fertilization
.

　　Researcher Miao Long, associate researcher Zhao Yanmei, and Dr.
Cao Zheng of the Institute of Biophysics, Chinese Academy of Sciences are the co-corresponding authors of this article.
Postdoctoral fellow Wang Qiushi, engineer Cao Zheng, doctoral student Du Baochen, and doctoral student Zhang Qi are the co-first authors of this article
.
Chen Lianwan, a senior engineer at the Institute of Biophysics, provided support for electron microscopy in this work, and the research was funded by the Ministry of Science and Technology and the National Natural Science Foundation of China
.